CircRNA_400029 promotes the aggressive behaviors of cervical cancer by regulation of miR-1285-3p/TLN1 axis.

Background: Cervical cancer (CC) is the highest incidence of female malignant tumor in China. Circular RNAs (circRNAs) has been reported to affect CC progression by altering mRNA stability at the transcriptional level or binding to miRNAs to produce competitive endogenous RNA (ceRNA). For this purpose, our study was aimed to investigate the function effects and the potential regulatory mechanism of the circRNA_400029 in CC cells. Materials and methods: The expression levels of circRNA_400029 and miR-1285-3p were detected by real time polymerase chain reaction (RT-PCR). Similarly, the mRNA and protein levels of TLN1 (Talin 1) was detected by RT-PCR and Western blot. Cell-Counting Kit-8 (CCK-8), EdU and Flow cytometry assay were used to detect cell proliferation, cell cycle and apoptosis. Then the Transwell assays were used to test cell migration and invasion. Besides this, the functional targets were confirmed by Dual luciferase reporter assays. Tumor xenograft in nude mice checked the result in vivo. Results: To begin with, circRNA_400029 was upregulated in CC cells and tissue. Knockdown circRNA_400029 inhibited cell proliferation, migration and invasion while induced cell apoptosis. Interestingly, miR-1285-3p targeted circRNA_400029 and down-regulated of miR-1285-3p could reverse the effects of circRNA_400029 weak-expression on progression and apoptosis of CC cells. Moreover, TLN1 was up-regulated in CC cells and identified as a direct target of miR-1285-3p. Meanwhile, we found that miR-1285-3p negatively regulated the function of TLN1. Finally, the circRNA_400029/miR-1285-3p/TLN1 axis could affect tumor growth in vivo. Conclusion: The overexpressed circRNA_400029 promoted CC proliferation, migration and invasion while deduced apoptosis by sponging miR-1285-3p to regulate TLN1. CircRNA_400029 was a potential onco-circRNA in CC, and might be a promised therapy target.

Clinical value of serum squamous cell carcinoma antigen levels in predicting chemosensitivity, lymph node metastasis, and prognosis in patients with cervical squamous cell carcinoma.

BACKGROUND: Our aim was to investigate the value of serum squamous cell carcinoma (SCC) antigen levels in predicting chemosensitivity, lymph node metastasis, as well as prognosis in patients with cervical squamous cell carcinoma who received neoadjuvant chemotherapy (NACT). METHODS: This retrospective study enrolled 103 patients with cervical squamous cell carcinoma and then compared the SCC antigen levels between patients who underwent NACT followed by radical surgery (NACT group) and those who underwent radical surgery alone (conventional group), and a correlation analysis between SCC antigen levels and chemosensitivity, lymph node metastasis, or survival time was conducted. RESULTS: The SCC antigen levels changed after NACT and were associated with chemosensitivity. Moreover, the optimal cut-off value of the percentage decrease in SCC antigen level after the first chemotherapy (FSCC (%)) was 42.0%, which could be used for assessment of chemosensitivity. The rate of positive lymph nodes in patients with pretreatment SCC antigen levels ≥3.9 ng/mL was significantly decreased after NACT. The overall survival (OS) of NACT group was significantly longer than that of conventional group when the pretreatment SCC antigen levels were ≥ 4.55 ng/mL. The OS and progression-free survival rates of patients with SCC antigen levels < 2.7 ng/mL were longer than those ≥2.7 ng/mL after the first chemotherapy. CONCLUSIONS: The 42.0% of FSCC (%) after NACT is a reliable indicator of chemosensitivity. Pretreatment and posttreatment SCC antigen levels can be used in evaluating the lymph node metastases and prognosis of patients with cervical squamous cell carcinoma.

Reduced microRNA-451 expression in eutopic endometrium contributes to the pathogenesis of endometriosis.

BACKGROUND: Endometriosis (EMs) is a chronic and recurrent, but benign, disease in women of reproductive age, and EMs patients have a high risk of developing gynecological tumors and autoimmune disorders. The etiology of EMs is not clear. Certain genetic markers in the eutopic endometrium are key in the pathogenesis of EMs. MicroRNAs (miRNAs) are implicated in several biological processes, such as cell proliferation, differentiation, and apoptosis. MiR-451 is interesting, as it acts as a tumor suppressor and is relevant to the poor prognosis of cancers. AIM: To evaluate the expression levels and role of miR-451 in the eutopic endometrium and predict possible targets of miR-451 and related signaling pathways. METHODS: Quantitative real-time polymerase chain reaction was used to evaluate miR-451 expression in cultured cell lines as well as in pathologic tissues from 40 patients with EMs and 20 donors with no history of the disease (controls). Cell Counting Kit-8 and flow cytometric assays were performed to determine cell proliferation and survival rates after transfection with miR-451 mimics and siRNAs. MiR-451 targets were predicted using miRDB and miRcode target-predicting databases. RESULTS: We observed lower miR-451 levels in eutopic endometrial tissues from patients with EMs than in control tissues, and this difference was not related to the American Society for Reproductive Medicine stage. Ectopic overexpression of miR-451 in eutopic cells induced apoptosis and inhibited cell proliferation. SiRNA-mediated miR-451 knockdown reversed these effects. Using miRDB and miRcode, we identified 12 potential miR-451 target genes. We hypothesize that the expression of YWHAZ, OSR1, TTN, and CDKN2D may be regulated by miR-451 and be involved in disease pathogenesis. CONCLUSION: Reduced miR-451 expression in the eutopic endometrium contributes to the pathogenesis of EMs by promoting cell proliferation and reducing apoptosis. Thus, miR-451 is a novel biomarker for EMs. YWHAZ, OSR1, TTN, and CDKN2D are potential target genes of miR-451 and may have key roles in this disease.

GATA-3 Stimulates Proliferation of Endometriotic Cells.

OBJECTIVES: This study aimed to investigate whether there was a correlation between the expression of GATA binding protein 3 (GATA-3) and the proliferation of endometrium cells from women with endometriosis. MATERIAL AND METHODS: Endometrial tissue was collected from 20 women with endometriosis (experimental group) and 10 women without endometriosis (control group). The primary endometrial cells were then cultured and identified. Each group of cells were further divided into four groups. The control group was divided into control-no treatment (C-N), control-negative control (C-NC, transfected with a scrambled siRNA negative control), control-lentiviral transfection control (C-LC) and control-GATA-3 lentiviral transfection (C-G3); the experimental group was divided into experiment-no treatment (E-N), experiment-negative control (E-NC), experiment-lentiviral transfection control (E-LC) and experiment-siGATA-3 lentiviral transfection (E-siG3). RT-PCR and western blot were used respectively to detect the endometrium cells' GATA-3 mRNA and protein expression. Further, Cell Counting Kit-8 was used to measure the endometrium cells' proliferation. RESULTS: The relative expression of GATA-3 mRNA and protein in the E-N group was significantly higher than in the D-N group and E-siG3 group (P < 0.05). The relative cell proliferation rate in the C-G3 group at 48 h, 72 h and 96 h was significantly higher than in the C-N, C-NC and C-LC groups (P < 0.05), while in the experimental group, the proliferation rate of the E-siG3 group was significantly lower than in the E-N, E-NC and E-LC groups (P < 0.05). CONCLUSIONS: This study suggests the role of GATA-3 in promoting proliferation of endometrial cells; therefore, GATA-3 may play an essential role in the development of endometriosis.

Accuracy of Physical Examination, Transvaginal Sonography, Magnetic Resonance Imaging, and Rectal Endoscopic Sonography for Preoperative Evaluation of Rectovaginal Endometriosis.

The aim of this study was to compare the effectiveness of physical examination, transvaginal sonography, magnetic resonance imaging, and rectal endoscopic sonography for the identification of rectovaginal endometriosis and potential rectal infiltration. Women with suspected rectovaginal endometriosis underwent physical examination, transvaginal sonography, magnetic resonance imaging, and rectal endoscopic sonography. Evaluation was performed for the presence of rectovaginal endometriotic foci and rectal infiltration. The findings obtained with these methods were compared with those of surgical and histopathological examination. Sensitivity, specificity, positive predictive values, and negative predictive values were evaluated for each method. Rectovaginal endometriosis was histologically confirmed in 21 (72.4%) of 29 women. With respect to diagnosis of rectovaginal endometriosis, the sensitivity, specificity, and accuracy of physical examination were 95.2%, 62.5%, and 86.2%; those of transvaginal sonography were 42.9%, 87.5%, and 55.2%; those of magnetic resonance imaging were 90.5%, 87.5%, and 89.7%; and those of rectal endoscopic sonography were 81.0%, 75.0%, and 79.3%, respectively. With respect to identification of rectal infiltration, the sensitivity, specificity, and accuracy of transvaginal sonography were 26.7%, 85.7%, and 55.2%; those of magnetic resonance imaging were 73.3%, 92.9%, and 82.8%; and those of rectal endoscopic sonography were 86.7%, 85.7%, and 86.2%, respectively. Magnetic resonance imaging combined with physical examination seem to be the main approach for the presurgical assessment of rectovaginal endometriosis. Rectal endoscopic sonography is a worthwhile method for the diagnosis of rectal infiltration.

Squamous cell carcinoma antigen expression in tumor cells is associated with the chemosensitivity and survival of patients with cervical cancer receiving docetaxel-carboplatin-based neoadjuvant chemotherapy.

The present study examined the association between squamous cell carcinoma antigen (SCCA) expression and the response of patients with cervical cancer to docetaxel-carboplatin (DC) combination chemotherapy, in order to determine the prognostic potential of SCCA expression. A total of 21 patients were enrolled with stage IB2 or stage IIA2 SCC. Of these, 9 patients had chemotherapy-sensitive cancer (2 cases with a complete response and 7 cases with a partial response) and 12 patients had chemotherapy-resistant cancer (12 cases of stable disease and no cases of progressive disease). Patients were treated with two cycles of DC chemotherapy (docetaxel 75 mg/m2 plus cisplatin 25 mg/m2) with 21-day intervals, followed by radical surgery. SCCA expression levels prior to and following chemotherapy were evaluated using immunohistochemistry. Following DC chemotherapy, the SCCA expression levels decreased in the patients with chemotherapy-sensitive cancer, but not in those with chemotherapy-resistant cancer (P=0.042). Significant survival differences between the SCCA-positive and -negative patients following chemotherapy (P=0.009) was observed. However, no statistically significant difference in survival between patients with chemotherapy-sensitive and chemotherapy-resistant cancer, or between patients with SCCA-positive and -negative expression prior to chemotherapy was observed. Overall, the chemotherapy sensitivity of patients with cervical cancer was associated with decreased SCCA expression levels following DC chemotherapy. Therefore, SCCA expression levels following DC chemotherapy may potentially be used in the clinical prognosis for cervical cancer patients who receive DC chemotherapy and subsequent radical surgery.

Evaluation of estrogen in endometriosis patients: Regulation of GATA-3 in endometrial cells and effects on Th2 cytokines.

AIMS: Endometriosis (EM) is a hormone-dependent chronic inflammatory disease, usually accompanied by a high level of localized estrogen and abnormal levels of cytokines, which are regulated by GATA-3 in lymphocytes. This study aimed to investigate the role of estrogen on GATA-3 expression and the relationship between GATA-3 and cytokine response. METHODS: Endometrial tissues collected from 20 patients who underwent laparoscopic or open surgery were used. Immunohistochemistry, quantitative polymerase chain reaction, Western blot analysis, cell transfection, estrogen treatments and enzyme-linked immunosorbent assays were performed to evaluate the effects of estrogen on GATA-3 expression and the relationship between estrogen-induced GATA-3 and the Th2 immune status of EM. RESULTS: Estrogen regulated the expression of GATA-3 in a dose and time-dependent manner. GATA-3 was relocated from the cytoplasm to the nucleus. Estrogen and GATA-3 regulated Th2 cytokine expression in eutopic endometrial cells, including interleukin (IL)-6, IL-8 and IL-10. Moreover, interferon-γ and IL-2 were highly expressed in the GATA-3 knockdown groups. CONCLUSION: In summary, GATA-3 was induced by estrogen and may promote the occurrence and development of EM by regulating the secretion of cytokines in the eutopic endometrial cells of EM patients.

[Relief of urinary retention after radical hysterectomy for cervical cancer patients by acupressure: a randomized controlled trial].

OBJECTIVE: To study whether acupressure could relieve urinary retention after radical hysterectomy in cervical cancer patients. METHODS: A randomized controlled prospective double-blinded trial was carried out in 107 urinary retention patients undergoing grade III radical hysterectomy. They were assigned to Group A (positive acupoints, 40 cases), Group B (negative acupoints, 32 cases) , and Group C (with no acupoints, 35 cases). All patients received protective 115 000 potassium permanganate sitz bath, 15 - 20 min each time, 3 times per day. Patients in Group A received acupressure at positive points [liniao point and Qihai (RN6)] combined points by syndrome typing [Guanyuan (RN4) , Zhongji (RN3) , Shenshu (BL23) , Zusanli (ST36), Sanyinjiao (SP6), and Taixi (K13)]. Patients in Group B received negative acupressure at sham-acupoints (for adjusting gastrointestinal functions). Patients in Group C only received conventional sitz bath. All medication was performed 3 times per day, 7 days as one therapeutic course, 21 days in total. The residual urine volume was detected. The recovery time for bladder function was recorded. The average residual urine volume was also recorded at day 7, 14, and 21. RESULTS: Compared with Group B and C, the time for ureter retention was shortened for mild and severe CKD patients in Group A (P <0. 01). The residual urine volume was also lessened for mild and severe CKD patients in Group A at day 7, 14, and 21 (P <0.01). CONCLUSION: Cervical cancer patients could relieve urinary retention by self-acupressure after radical hysterectomy.

Inducing malignant transformation of endometriosis in rats by long-term sustaining hyperestrogenemia and type II diabetes.

This study aimed to induce malignant transformation of endometriosis in Sprague-Dawley rats by hyperestrogenemia and type II diabetes and evaluate its similarity with human disease in biological features. Rats with surgically induced endometriosis were randomized into two groups: those treated with estradiol (5 mg/kg three times/week after surgery), streptozotocin (25 mg/kg, 1 month after surgery), and high carbohydrate-and-fat feed (Es group); and those treated with placebo saline and standard feed (control group). All rats were randomly killed 2, 4, or 8 months after surgery. The endometriosis lesions and the corresponding eutopic endometria were subjected to morphological evaluation, TUNEL, and immunohistochemical analysis for the expressions of proliferating cell nuclear antigen, phosphatase and tensin homolog, phosphorylated protein kinase B, and phosphorylated mammalian target of rapamycin proteins. In the Es group, three cases (6.0%) of endometriosis showed atypical hyperplasia accompanied by simple hyperplastic eutopic endometria, and two cases (4.0%) of endometriosis showed endometrioid carcinoma accompanied by atypical hyperplastic eutopic endometria. In the Es group, the activity of organelles and the expressions of proliferating cell nuclear antigen, phosphorylated protein kinase B, and phosphorylated mammalian target of rapamycin increased, and the level of phosphatase and tensin homolog and TUNEL positivity decreased progressively in the order of endometriosis, atypical endometriosis, and malignant endometriosis. The same tendency was found in the corresponding eutopic endometria. The induced malignant endometriosis showed similarities with human disease in the pathological process and histomorphological and molecular biological features. The method is feasible. The malignant transformations of endometriosis and eutopic endometria may have correlations and similarities, but the former may suffer a higher risk of canceration.

Transvaginal laparoscopic appendectomy simultaneously with vaginal hysterectomy: initial experience of 10 cases.

BACKGROUND: Natural orifice transluminal endoscopic surgery (NOTES) involves the introduction of instruments through a natural orifice into the peritoneal cavity to perform surgical interventions. The vagina is the most widely used approach to NOTES. We report the utilization of the vaginal opening at the time of vaginal hysterectomy as a natural orifice for laparoscopic appendectomy. MATERIAL AND METHODS: We reviewed cases of 10 patients with chronic appendicitis who underwent transvaginal laparoscopic appendectomy simultaneously with vaginal hysterectomy. A laparoscopic approach was established after removal of the uterus, and the appendix was removed transvaginally. Among the 10 cases, 5 were conducted under gasless laparoscopy by using a simple abdominal wall-lifting instrument. RESULTS: All procedures were performed successfully without intraoperative or major postoperative complications. The appendectomy portion of the procedure took approximately 21 minutes to 34 minutes. All patients were discharged less than 4 days after surgery, without external scars. CONCLUSIONS: Transvaginal appendectomy with rigid laparoscopic instruments following vaginal hysterectomy appears to be a feasible and safe modification of established techniques, with acceptable outcomes.

Identification of differentially methylated genes in the malignant transformation of ovarian endometriosis.

BACKGROUND: Key roles for epigenetic mechanisms in tumorigenesis are well accepted, while the relationship between gene methylation and malignant transformation of ovarian endometriosis (EMS) was seldom reported. In this study, we aimed to screen for aberrantly methylated genes associated with the malignant transformation of ovarian EMS and to preliminarily verify the reliability of screened results by detecting the methylation status and protein expression of the candidate gene in a larger scale of formaldehyde-fixed and paraffin-embedded (FFPE) samples. METHODS: Methylated CpG island amplification coupled with representational difference analysis (MCA-RDA) was performed on 3 couples of endometriosis-associated ovarian carcinoma (EAOC) fresh samples to identify differentially methylated candidate genes related to malignant transformation of ovarian EMS; Methylation-specific PCR (MSP) and immunohistochemistry were performed in 30 EAOC samples to detected the methylation status and protein expression of RASSF2 gene to verify the reliability of MCA-RDA results. RESULTS: Nine differentially methylated genes were obtained by MCA-RDA as candidate genes for malignant transformation of EMS; Methylation frequency of RASSF2 in the neoplastic tissues of EAOC group was higher than that in the ectopic endometria (p < 0.05). While protein expression of RASSF2 in the neoplastic tissues was lower than that in the ectopic endometria of the EAOC group (p < 0.05) Absence of protein expression of RASSF2 was significantly correlated with the promoter methylation of the gene (p < 0.05). CONCLUSIONS: RASSF2, RUNX3, GSTZ1, CYP2A, GBGT1, NDUFS1, SPOCK2, ADAM22, and TRIM36 were candidate genes for malignant transformation of ovarian EMS and epigenetic inactivation of RASSF2 by promoter hypermethylation is an early event in malignant transformation of ovarian EMS. The screen results were reliable and worthy of further study.

Pure NOTES transvaginal appendectomy with gasless laparoscopy.

BACKGROUND: The vagina is the most widely used approach to natural orifice transluminal endoscopic surgery. However, a gas leak can significantly affect transvaginal operations during pneumoperitoneum laparoscopy. We tried to establish the proper technique for transvaginal appendectomy under gasless laparoscopy. MATERIALS AND METHODS: Five patients with chronic appendicitis were selected to receive gasless laparoscopic transvaginal appendectomy with concurrent vaginal hysterectomy. An abdominal wall-lifting device was applied after removal of the uterus, and the appendix was removed transvaginally. Clinical data such as operative duration, bleeding volume, morbidity, and hospital stay duration were analyzed. RESULTS: All procedures were performed successfully, without intraoperative or major postoperative complications. The appendectomy portion of the procedure took approximately 20-30 minutes, with minimal blood loss. All patients were discharged, scar-free, 3 d after surgery. CONCLUSIONS: Transvaginal appendectomy with gasless laparoscopy after vaginal hysterectomy appears to be a feasible and safe modification of established techniques, with acceptable outcomes.

Evaluation of the transvaginal resection of low-segment cesarean scar ectopic pregnancies.

OBJECTIVE: To evaluate the transvaginal approach for exogenous cesarean scar ectopic pregnancy (CSEP) lesion resection, according to our experience. DESIGN: Retrospective study. SETTING: University hospital. PATIENT(S): Twenty-three patients with confirmed exogenous CSEP treated with transvaginal resection of the lesions at the Shengjing Hospital of China Medical University. INTERVENTION(S): Lesion resection by the transvaginal approach. MAIN OUTCOME MEASURE(S): Surgery time, intraoperative blood loss, postoperative drainage volume, and average days of postoperative hospitalization were compared between the transvaginal procedure group and the transabdominal procedure group. RESULT(S): Significant differences were found. No significant differences were found in the drop of serum hCG level after 48 hours and the postoperative recurrence rate between the two groups. The rate of successful treatment was 96%. CONCLUSION(S): The transvaginal approach for exogenous CSEP lesion resection possesses many advantages. In the hands of a skilled surgeon it can readily address the treatment problems posed by an exogenous CSEP without imposing an increase in surgical risk or technical difficulty.

Silencing Cofilin-1 blocks PDGF-induced proliferation in eutopic endometrium of endometriosis patients.

Platelet-derived growth factor (PDGF) is well known to induce proliferation in variety of cells. The aim of this study was to investigate whether there is a correlation between the expression of Cofilin-1 (CFL1) and the proliferation of eutopic endometrium stromal cells (ESC) in response to PDGF stimulation. Results show that PDGF induced the expression of CFL1 in ESC and promoted proliferation of ESC significantly in a time- or dose-dependent manner. After silencing CFL1, the effect of PDGF on promoting ESC proliferation was significantly decreased. These data demonstrate that PDGF can induce CFL1 expression in ESC. Silencing CFL1 blocks PDGF-induced proliferation in ESC.

[Transvaginal appendectomy with traditional laparoscopic tools].

OBJECTIVE: To establish the technique and methodology of appendectomy through transvaginal natural orifice transluminal endoscopic surgery(NOTES)under laparoscopy. METHODS: Three cases of chronic appendicitis were selected to receive laparoscopic transvaginal resection of appendixes with concurrent vaginal hysterectomies for uterine fibroids at Shengjing Hospital of China Medical University from November 2010 to November 2011. The procedure was performed by a multidisciplinary team composed of surgeons and gynecologists. The clinical data such as operative duration, bleeding volume, morbidity and stay duration were collected and analyzed. RESULTS: Appendectomies were performed with the working laparoscopic tools inserted through vagina. The en bloc resection was removed transvaginally through laparoscope. It took 34, 23 and 26 minutes respectively to complete the appendectomies. And the blood loss volume was minimal. There were no postoperative complications. And the patients were discharged scar-free after 3 days. CONCLUSIONS: Transvaginal appendectomy is both feasible and safe when performed by a multidisciplinary team. As an emerging innovation, natural orifice transluminal endoscopic surgery is mini-invasive, better tolerated and more respectful of esthetics.

Detecting and investigating the significance of high-frequency LOH chromosome regions for endometriosis-related candidate genes.

To detect the high-frequency loss of heterozygosity (LOH) chromosome regions for ectopic endometrium of ovarian endometriosis (EMs) and to investigate the significance of high-frequency LOH chromosome regions in EMs, we obtained ectopic endometrium by laser capture microdissection (LCM (22 samples)), manual capture microdissection (MCM (18 samples)), and routine dissection (14 samples), respectively. After restriction and circularization-aided rolling circle amplification (RCA-RCA), LOH was detected at 12 microsatellite (MS) loci. The frequency of LOH was 59.09% (13/22) in LCM group, 61.11% (11/18) in the MCM group and 21.43% (3/14) in the routine dissection group. The latter was significantly lower when compared with the former two (p < 0.05). In the LCM group, candidate chromosome regions 17q21.31 and 9p21.3 had LOH frequencies of 23.8 and 13.6%, respectively. The highest LOH frequency was detected at the locus AAAT2 on chromosome 17q21.31 (40%). The chromosome region with the highest frequency of LOH for ectopic endometrium was 17q21.31, especially at the AAAT2 locus, which prompted that down regulation of the candidate genes nearby the locus might be one of the mechanisms of EMs pathogenesis. LCM combined with RCA-RCA is a reliable technique for analyzing endometrial LOH at multiple MS loci. MCM combined with RCA-RCA, which provided similar results, was more cost-effective.

Expression of miR-126 and Crk in endometriosis: miR-126 may affect the progression of endometriosis by regulating Crk expression.

PURPOSE: To evaluate the relationship between miR-126 and Crk and discuss the role of miR-126 in the development and progression of endometriosis (EMs). METHODS: The expression levels of miR-126 and Crk mRNA were quantified using real time fluorescent quantitative polymerase chain reaction (real time PCR) in ectopic endometrium (ECs) and eutopic endometrium (EUs) in patients with EMs and normal endometrium (ENs) in EMs-free subjects. The expression levels of Crk protein in all samples were evaluated by Western blot. RESULTS: The expression level of miR-126 was significantly downregulated in ECs versus EUs (p = 5.45E(-5)) in the experimental group and in EUs versus ENs (p = 0.019). The expression level of Crk mRNA did not distinguish ECs from EUs (p = 0.995) but was overexpressed in EUs versus ENs (p = 0.006). Crk protein was overexpressed in ECs versus EUs (p = 0.002) in the experimental group and in EUs versus ENs (p = 1.13E(-6)). The expression level of miR-126 had no correlation with Crk mRNA (p = 0.496) but was negatively correlated with Crk protein (p = 3.134E(-5)). The expression level of miR-126 in EUs and ECs was negatively correlated with American Fertility Society (AFS) stage (p = 0.022, p = 0.025) and AFS score (p = 0.002, p = 0.007). miR-126 expression decreased with the progression of EMs, but the decrease was not significantly different. CONCLUSIONS: miR-126 may play an initial role in the development and progression of EMs. Crk may be regulated by miR-126, and synergism between abnormal expressions may play an important role in the pathogenesis of EMs.

[Epigenetic inactivation of SPOCK2 in the malignant transformation of ovarian endometriosis].

OBJECTIVE: To investigate epigenetic inactivation of SPOCK2 gene in the malignant transformation of ovarian endometriosis (EM) by comparing the methylation status and protein expression of SPOCK2 gene in the malignant tissues, ectopic endometria and the eutopic endometria of endometriosis. METHODS: From Jan. 2005 to Jan. 2011, 22 paraffin-embedded specimens diagnosed as malignant transformation of ovarian endometriosis (EAOC) including 11 cases with ovarian endometrioid carcinoma, 8 cases with clear cell carcinoma, 2 cases with serous cystadenocarcinoma and 1 case with mucous cystoadenocarcinoma matched with 22 cases with ovarian endometriosis and 16 cases with normal endometrium form cervical intraepithelial neoplasia(CIN) patients as controls in Department of Obstetrics and Gynecology of Shengjing Hospital. Twenty-two malignant tissues, 15 ectopic endometria and 10 eutopic endometria were captured by microdissection in EAOC group; 22 ectopic endometria and 17 eutopic endometria were captured in EM group; 22 endometrium were captured in the NE group. The methylation statue of SPOCK2 was determined by combined bisulfite restriction analysis, and the protein expression of SPOCK2 was evaluated by immunohistochemistry. RESULTS: (1) Methylation of SPOCK2: in the EAOC group, the frequency of SPOCK2 hypermethylation in malignant tissue was 45% (10/22), which was significantly higher than 1/15 in the ectopic endometrium (P < 0.05). There was no statistical difference of the frequency of SPOCK2 hypermethylation in ectopic endometrium in the EAOC group (1/15) and EM group (5%, 1/22) (P > 0.05). (2) SPOCK2 protein: the loss rate of SPOCK2 was 44% (11/22) in malignant tissue in EAOC group, which were significantly higher than 2/15 of in ectopic endometrium of EAOC (P < 0.05). However, there was no remarkable difference in loss rate of SPOCK2 protein between ectopic endeometrium of EAOC and endometrium of EM [2/15 vs. 5% (1/22), P > 0.05]. No significantly difference in loss rate of SPOCK2 in eutopic endometrium was observed among three groups (P > 0.05). (3) The abnormal methylation of SPOCK2 could lead to loss expression of protein (P < 0.05). CONCLUSION: Epigenetic inactivation of SPOCK2 gene is involved in the malignant transformation of ovarian endometriosis.

Silencing of cofilin-1 gene attenuates biological behaviours of stromal cells derived from eutopic endometria of women with endometriosis.

BACKGROUND: Eutopic endometria with endometriosis (EMs) differ dramatically from normal endometria, physiologically and biochemically, yet the pathogenesis of EMs remains unclear. Cofilin-1 (CFL1), a critical modulator of the actin cystoskeleton, is associated with tumour progression, cell motility, cell adhesion, cell invasion and angiogenesis. Although eutopic endometria with EMs exhibit many malignant-like behaviours and a higher expression of CFL1 than normal endometria, the effects of CFL1 on the pathogenesis of EMs are unknown. The aim of this study was to explore the role of CFL1 expression in proliferation, apoptosis, adhesion, invasion, angiogenesis and ultrastructure of endometrial cells. METHODS: We isolated and cultured stromal cells derived from the eutopic endometria of 30 patients with advanced ovarian EMs (ESCs, Stromal Cells of eutopic endometria in Endometriosis patients) and 30 control patients without EMs (NSCs, Stromal Cells of eutopic endometria in Non-endometriosis patients), and evaluated their proliferation, apoptosis, adhesion, invasion and expression of markers of adhesion, invasion and angiogenesis in vitro. In addition, these functions were examined after short hairpin RNA (shRNA) was used to silence the CFL1 gene in ESCs, and pEGFP-N1-CFL recombinant plasmid was transiently transfected into NSCs to up-regulate CFL1 expression. RESULTS: Under basal conditions, CFL1 mRNA and protein were overexpressed in ESCs. Proliferation, adhesion, invasion and markers of adhesion, invasion and angiogenesis were enhanced in ESCs compared with NSCs; in contrast, the apoptosis rate was lower in ESCs than in NSCs. Silencing the CFL1 gene in ESCs markedly attenuated proliferation, adhesion, invasion and expression of the markers, but enhanced apoptosis. Conversely, up-regulation of CFL1 in NSCs increased proliferation, adhesion, invasion and expression of the markers but reduced apoptosis. CONCLUSIONS: The overexpression of CFL1 in ESCs is associated with enhanced proliferation, adhesion, invasion and angiogenesis and reduced apoptosis in EMs. These malignant-like behaviours of ESCs in EMs can be attenuated by inducing CFL1 gene silencing with shRNA interference.

[Relationship between cofilin-1 expression and implantation capacity in eutopic endometrium of patient with endometriosis].

OBJECTIVE: To investigate the relationship between expression of cofilin-1 and in vivo implantation capacity of eutopic endometrium of endometriosis. METHODS: Eutopic endometrium of 20 cases with stage III or IV endometriosis were obtained by laparoscopic or laparotomy surgery (endometriosis group) matched with 20 cases of eutopic endometrium from patients with cervical cancer in situ (control group) in Department of Obstetrics and Gynecology, ShengJing Hospital. All cases' eutopic endometrium were collected and injected into abdominal cavity of nude mice to establish endometriosis animal model, then the successful rate of animal model and volume of endometriosis lesion were calculated. The expression and positive rate of cofilin-1 protein were measured by western blot and immunohistochemistry staining. RESULTS: The mean volume of endometriosis lesions (2.38+/-0.22) mm3 in endometriosis group was significantly bigger than (0.36+/-0.08) mm3 in control group (P<0.05). The successful rate of establishing endometriosis model was 95% (19/20) in endometriosis group and 5% (1/20) in control group, which reached statistical difference (P<0.05). The expression and positive rate of cofilin-1 protein in eutopic endometrium and the expression of cofilin-1 protein in endometriosis lesion of animal model were 0.82+/-0.06, 90% (18/20), 0.85+/-0.03 and 0.21+/-0.03, 20% (4/20), 0.22+/-0.02 in control group, which reached statistical difference (P<0.05). The successful rate of establishing endometriosis model with positive cofilin-1 in endometrium 86% (19/22) was significantly higher than 6% (1/16) of model's endometrium with negative cofilin-1 expression (P<0.05). CONCLUSIONS: Implanting capacity and cofilin-1 expression level of eutopic endometrium of endometriosis were more intensive than that of normal endometrium. High cofilin-1 expression was probably related with implanting capacity of eutopic endometrium of endometriosis.