Genome Wide Association Study And Functional Analysis Of Susceptibility Genes Of Gynecology Complex Disease

Background and objective:Endometriosis is one of common disease, which incidence is rising recently. It has been linked to infertility, pelvic pain and dysnebirrhea. However, there is little mechanism available about the pathogenesis. It is well known that endometriosis perform some malignant behavior such as invasive, distance metastasis, down-regμlation of apoptosis, up-regμlation of proliferation, recurrence. Additionally, it has risk of development of ovary cancer. Without clues researchers have preferred some oncogene as candidate gene for pathogenesis study, such as COX-2, TNF and FASLThe HGP and gene chip make it possible to find out the susceptible gene without hypothesis. Some common diseases, diabetes for instance, have been studied in genome wide with microarray, and susceptible makers and genes have been discovered. In this study we performed genome wide screening to find out the gene associated with endometriosis and analyzed the mechanism involved. Methods:1 100 endometriosis specimens and 100 control cases were collected, the genome DNA were used for Affymetrix SNP 6.0 array analysis.2 Silhouette and adjusted t-test were used to pick out the SNPs associated with endometriosis. Then the genes has linkage with the SNPs were searched.3 Immunohistochemistry and western bolt were used to test the expression of MEIS1 in endometrium of specimen and control cases.4 Lentivirus vector of shMEIS 1 was constructed, which were transfected into endometrial cells. MTT assay and FCM assay were used to test the proliferation and apoptosis of endometrial cells.Results:1 We got 16 SNPs with both Silhouette and adjusted t-test, which were used as candidate locus. Then the genes has linkage with the SNPs were searched. MEIS1 was selected for further functional study.2 Immunohistochemistry and western bolt showed that the expression of MEIS1 was weaker in endometrium of endometriosis than that in control cases, this res u It coμId be obtained in P2 P3 S1 S2 stages.3 Knock down the expression of MEIS1 in endometrial cells coμId enhance of proliferation and down-regμlate of apoptosis.Conclusions:The susceptibility gene MEIS1 down-regμlated in endometrial of endometriosis, anddecreased the apoptosis and played role in pathogenesis of endometriosis. Background and objective:Cervical cancer, the second most frequently diagnosed malignancy in women worldwide, is a mμlti-step process disease, developing through stages designated cervical intraepithelial neoplasia (CIN), CIN I,CINⅡand CIN III, eventually to invasive carcinoma stage. Despite the generally good prognosis for early-stage cervical cancer, approximately one-third of these patients are expected to ultimately die of the disease resulting from metastasis and recurrence. The identification and functional characterization of molecules involved in development, metastasis and outcome, exploitation of candidate targets for diagnosis and therapy, therefore, are of importance for avoiding the threat of cervical cancer. Integration of HPV genome DNA into the host genome is a major mechanism of cervical carcinogenesis. Over 100 types of HPV virus have been described, and based on their association with clinical disease, HPVs have been categorized into low-risk, intermediate-risk and high-risk type. High-risk HPVs are thought to lead to carcinogenesis of cervical cancer throμgh binding and inactivation of tumor suppressors, p53 and retinoblastoma protein (Rb), by the viral-transforming protein E6 and E7 respectively. However, there shoμld be some mechanism unknown involved. Such as senescence, epithelial mensenchymal transition, stem cell. And, the gene involved cervical cancer coμld be also associated with the genetic background of host genome, for that, candidate gene approach has been used to identify the gene of pathogenesis in last years. Nowadays, HGP and microarray provided methods for screening associated gene in genome wide, which coμld be used for future functional study. Methods: 1 142 archival formalin-fixed and paraffin-embedded cervix specimens were obtained from the Department of Pathology. There were 14 cases of normal cervical tissues,58 cases of cervical intraepithelial neoplasia (CIN) and 70 cases of (宫颈癌). Tissues were subjected to TWIST, CBX3 and E-cadherin immunohistochemical staining and statistical analysis. Statistical analysis was performed using SPSS (SSPS Inc., USA) 13.0 software. Differences in TWIST, CBX3 and E-cadherin expression from the five histological groups of specimens were analyzed using the non-parametric Kruskal-Wallis test and Mann-Whitney test, where applicable. Pearson chi-square test and the Fisher’s exact test (wherever was applicable) were used to compare clinical characteristics of patients with or without lymph node metastasis and the TWIST expression. Logistic regression models were fit to identify the factors significantly correlated with TWIST expression levels and the risk of developing metastasis.2 The express vectors of TWIST1/2, siTWIST1/2 were constructed, which were used to transfected into END, ECT, Siha, C33a cell lines. The EMT and senescence phenotype were observed using microscope. SA-β-gal staining was used to test senescence phenotype. Immunofluorescence was used to test E-cadherin expression so that analysis the EMT program.3 Western blot, Confocal, MTT, FCM, Transwell assay, Wooden assay were used to study the role of TWIST in carcinogenesis and invasion and metastasis of cervical cancer in vitrol.Results:1 TWIST1/2 were express in both cytoplasm and nucleus. The cytoplasmic and nuclear expression of TWIST were up-regμlated during the process of carcinogenesis of cervix (all P<0.05). Significant differences were found in both the cytoplasmic and nuclear (all P<0.05) staining of TWIST between the subgroups with or without lymph node metastasis. The staining of CBX3 showed most strong in stage of CINⅠand CINⅡ.when the leision developed,the expression of CBX3 dcreased in CINⅢand invasive tumor. The specimens that showed low expression of TWIST were also showed significant E-cadherin membrane staining, in contrary, when TWIST were up-regulated, E-cadherin was more cytoplasmic staining and membranous staining was weak. When age, the size of tumor, differentiation, cytoplasmic TWIST2, nuclear TWIST2 and aberrant E-cadherin expression were entered into the logistic regression model, only the high cytoplasmic TWIST2 expression (P=0.001, OR=6.527,95%CI 1.928-22.093), showed a significantly higher risk of developing metastatic lesions.2 The cells transfected with express vectors of TWIST 1/2, showed mensenchymal phenotype, and the E-cadherin transfer from member to cytoplasm. Meanwhile, the cells transfected with siTWIST1/2 showed senescence phenotype, and showed stronger staining of SA-β-gal.3 Down-regμlation of TWIST induced apoptosis in Siha and C33a cells. Meanwhile, down-regμlation of TWIST decrease the proliferation, and increase the ability of invasive and metastasis of cervical cancer cell lines.Conclusion:The transcription factor TWIST up-regμlated during the tumorigenesis and progression of cervical cancer. The overexpression may correlate with overgoing of senescence and induction of EMT, which may induce the malignance behavior of cervical cancer. Objective To explore the sensitivity and the molecular mechanism of cisplatin-resistance ovarian cancer cell line C13 to proteasome inhibitors and the combination with cisplatin.Methods After different treatments, methyl thiazolyl tetrazolium (MTT) assay was applied to examine the cell viability, annexin-Ⅴ/propidium iodide (PI) apoptosis detection kit was used to determine the apoptosis rate of different groups, western blotting assay was introduced to evaluate the expression levels of(Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein (cFLIPs), and the activity of caspase-8 was examined.Results MTT assay shown that the cell viability ratios of combination group at serial time points from 12 h to 72 h were (56.0±8.4)%, (44.7±7.3)%, (33.7±11.2)%, (27.6±8.0)%, (27.6±7.6) and (28.1±2.4)%, which were much lower than those of cisplatin group (P<0.05). After treated for 24 h, apoptosis rates of cisplatin group, bortezomib group and combination group were (16.7±1.7)%, (23.4±2.1)% and (26.9±1.6)%,respectively. The rate of combination group was much higher than that of non-treated group and that of cisplatin group or bortezomib group (P<0.05). Western blot assay showed the changes of expression levels of cFLIPs, which were down-regulated seriously after cisplatin bortezomib or combination treatment [(43.2±2.3)% vs (75.7±3.0)% vs (67.9±2.1) %,P<0.05]。The caspase-8 activity of combination group was 5.6±1.6 folds than that of non-treated group, which was higher than those of other two groups [(2.3±1.0) and (4.2±0.9) folds,P<0.05].Conclusions The tumor cell lethal effect of cisplatin could be increase significantly by the combination application of proteasome inhibitors, bortezomib. And the cFLIPs/caspase-8 signaling pathway may be play an important role in the molecular mechanism of the combination treatment.研究背景蛋白酶体抑制剂是新型抗癌药物,单独或与其他抗癌药物联合使用时均可有效地促进肿瘤细胞凋亡,克服化疗耐药,增强化疗敏感性。硼替佐米[bortezomib;其他名称：万珂(velcade)]是第1个被美国食品与药品管理局(FDA)批准应用于治疗复发或耐药性多发性骨髓瘤的蛋白酶体抑制剂,并已显示出良好的疗效。体外研究显示,硼替佐米对多种恶性肿瘤细胞均具有良好的抑制作用,但在卵巢上皮性癌(卵巢癌)治疗方面的研究至今报道很少。因此,本研究观察了硼替佐米与顺铂单独及联合作用于卵巢癌细胞株C13细胞的效果,并对其诱导细胞凋亡的分子学机制进行探讨,为临床用于治疗卵巢癌提供理论依据。材料与方法一材料1.试剂：RPMI1640培养基为美国Gibco公司产品,新生牛血清为杭州四季青生物工程材料有限公司产品。硼替佐米为美国Millennium制药公司产品,用注射用水配成2.6 mmol/L溶液备用,作用细胞时终浓度为50 nmol/L；顺铂冻干粉剂为山东省德州制药厂产品,用注射用水配成2mmol/L溶液备用,作用细胞时终浓度为40nmol/L。膜联蛋白(annexin V)-碘化丙啶(PI)双染色凋亡检测试剂盒购于南京凯...