家蚕卵巢肿瘤基因Bmotu及其可变剪接

已知卵巢肿瘤基因(ovarian tumor gene,otu)在果蝇(Drosophila melanogaster)卵巢发育过程中发挥极其重要的作用,该基因突变会扰乱卵子形成的正常过程。为探究家蚕(Bombyx mori)是否具有类似果蝇生殖发育相关基因otu的同源基因,以及基因存在的可变剪接形式,在电子克隆的基础上,应用RT-PCR从家蚕精巢组织中获得了4条不同长度的Bmotu cDNA片段(GenBank登录号:HQ831341,HQ831342,HQ999998,HQ831343),其中3条由于无义突变导致翻译提前终止;而从卵巢组织中仅获得了1条Bmotu cDNA片段,该片段序列与精巢中扩增的登录号为HQ831343片段序列的5'端相同。结果表明Bmotu基因存在可变剪接,且雌、雄之间的剪接方式可能存在差异。生物信息学分析发现Bmotu基因编码蛋白与果蝇Otu的结构类似,含有半胱氨酸蛋白酶结构域(Otu结构域)、Tudor结构域、脯氨酸基序。研究结果有助于进一步探讨家蚕生殖发育机制。

卵巢上皮性癌BRCA1基因mRNA原位杂交检测的研究

目的 :研究在上皮性卵巢癌中BRCA1基因mRNA的表达情况及与病理参数的关系。方法 :采用原位杂交法检测 5 0例卵巢上皮性癌组织中BRCA1基因mRNA的表达情况。结果 :BRCA1mRNA的阳性表达率在卵巢癌组织、良性肿瘤组织及正常组织中分别为 2 4 %、81 8%和 10 0 % ,癌组织中BRCA1基因的表达率显著低于良性肿瘤及正常组织 ,P <0 0 1。随着卵巢癌分化程度的降低 ,BRCA1基因表达缺失增多 ,P <0 0 5 ;同时有淋巴结转移的癌组织 ,BRCA1表达减少。结论 :BRCA1基因mRNA表达的减少可能与上皮性卵巢癌的发生发展有关 ,同时BRCA1mRNA表达减少提示预后较差。

HLA-DR表达与卵巢癌临床病理及预后的关系

目的:探讨HLA-DR与卵巢癌临床病理参数及预后的关系。方法:应用免疫组化SP方法检测卵巢癌、卵巢良性上皮肿瘤及正常卵巢组织中HLA-DR的表达。结果:卵巢癌组织中HLA-DR阳性率为57.89%(33/57),显著高于正常卵巢组织及良性上皮肿瘤(P<0.05),早期卵巢癌HLA-DR的阳性率显著高于晚期(P<0.01);高分化组HLA-DR的阳性率显著高于中、低分化组(P<0.05);HLA-DR阳性率与肿瘤组织局部淋巴细胞浸润程度呈正相关(P<0.05)。结论:①HLA-DR异常表达可能与卵巢癌的发病机制有关;②HLA-DR的低表达预示卵巢癌恶性程度高。

The Relationship between Methylation and Expression Defect of Tumor Suppressor Gene p16INK4A in Epithelial Ovarian Cancer

Objective： To evaluate the expression of p16INK4A gene in ovarian cancer and analyze the relation between this alteration and the promoter methylation of p16INK4A DNA. Methods： Seven ovarian cancer cell lines and eighteen ovarian cancer specimens were selected for the study. Genomic DNA and RNA were extracted from fresh tissues and cell lines, DNA was treated with sodium bisulfite and then analyzed with methylation-specific PCR （MSP） to detect p16INK4A methylation. The expression of p16INK4A mRNA was detected by reverse transcription-polymerase chain reaction （RT-PCR）. In addition, the proliferation of methylated cell lines before and after treatment of demethylating agent 5-Aza-2＇-deoxycytidine （5-ADC） was examined with 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay in vivo. Results： Compared with the control, the expression of p16INK4A mRNA decreased significantly or absolutely defaulted in 10 of 18 （55.56%） ovarian cancer specimens and 71.4% （5/7） ovarian cancer cell lines （P〈0.05）, and the expression of p16INK4A protein also decreased （P〈0.05）. The decrease of p16INK4A was due, in part, to p16INK4A methylation, which was found in the first exon of three cell lines and six ovarian cancer specimens and the rate was 42.86% and 33.33% in ovarian cancer cell lines and specimens respectively. All the methylated cells and tissues showed expression defect of p16INK4A, but the treatment of 5-ADC reactivated the expression of p16INK4A in methylated cells and decreased the proliferation of tumor cells in vitro and in vivo. Conclusion： The expression defect of p16INK4A gene possibly has an important role in the development of ovarian cancer, and this alteration is due, in part, to the methylation of the first exon in p16INK4A.

THE ASSOCIATION OF THE EXPRESSION OF MTA1, NM23H1 WITH THE INVASION, METASTASIS OF OVARIAN CARCINOMA

Objective. To understanding the molecular mechanisms in invasion andmetastasis of the ovarian carcinoma, we investigate a novel candidate metastasis―associated gene (MTA1) and nm23Hl mRNA expression and mutation in ovarian carcinoma. Methods. Twenty primary ovariancarcinoma specimens, 20 corresponding lymph nodes and 8 normal ovarian was examined for mRNAexpression and mutation of MTA1 and nm23Hl genes by reverse-transcription ploymerase chain reaction(RT―PCR) and RT―PCR―SSCP analysis. The level of the expression was determined by the relativeoptic desity (ROD) of the PCR products. Results. The frequency of MAT1 overexpression was 100% (7/7)in primary ovarian carcinoma with metastasis but only 38.5% (5/13) in those without metastasis(P=0.0103). Overexpression of MAT1 was observed in 87.5% (6/7) of lymph nodes with metastasis butonly 23% (3/13). of lymph nodes without metastasis (P=0.0118). In contrast with MAT1, low expressionof nm23H1 mRNA was seen in 7 of 7 o-varian carcinoma with metastasis but only in 4 of 13(30%) ofthose without metastassis (P=0.0043). Low nm23H1 expression was also seen in 7 of 7 lymph nodes withmetastasis but only in 5 of 13 (38.5%) nonmetastatic lymph nodes (P=0.0102). The ROD ratio of MAT1to nm23Hl increased with the development of metastasis. No mutation of MAT1 and nm23H1 genes wasfound by SSCP analysis. Conclusion. The mRNA expression of MTA1 and nm23H1 is positively andnegatively correlated with lymph node metastasis, respectively. Expression abnormalities but notmutation of the two genes are frequent events related to lymph node metastasis of ovarian cancer.

EFFECTS OF RETINOIC ACID ON PROLIFERATION AND DIFFEREN-TIATION OF A HUMAN OVARIAN CARCINOMA CELL LINE:3AO

Objective To observe the effects of retinoic acid (RA) on the proliferation and differentiation of a human ovarian carcino-ma cell line: 3AO cells. Methods 3AO cell proliferation was evaluated by viable cell count, percentage of cells in each cycle phase were analyzed by flow cytometric analysis, alkaline phosphatase (AKP) activity was determined as described , and CA125 expression was measured by ELISA. Results RA could inhibit the proliferation of 3AO cells accompanied with morphological changes in a dose-dependent manner. Cell cycle analysis indicated that RA inhibition of 3AO cells growth occurred through induction of G1 arrest with a concomitant reduction in the proportion of cells in S phase, AKP activity increased significantly after treatment with RA(0.1 μmol/L) for 1-5 days. Dose-response studies revealed that the AKP activity increased to a different extent as a function of RA concentrations. Furthermore, RA could suppress the expression of CA125 tumor marker in 3AO cells.Conclusion RA could markedly inhibit the proliferation and induce the differentiation of 3AO cells. for 1-5 days. Dose

MicroRNA: a new and promising potential biomarker for diagnosis and prognosis of ovarian cancer

Epithelial ovarian cancer(EOC) is the leading cause of death among all gynecological malignancies. Despite the technological and medical advances over the past four decades, such as the development of several biological markers(mRNA and proteins biomarkers), the mortality rate of ovarian cancer remains a challenge because of its late diagnosis, which is specifically attributed to low specificities and sensitivities. Under this compulsive scenario, recent advances in expression biology have shifted in identifying and developing specific and sensitive biomarkers, such as micro RNAs(miRNAs) for cancer diagnosis and prognosis. MiRNAs are a novel class of small non-coding RNAs that deregulate gene expression at the posttranscriptional level, either by translational repression or by mRNA degradation. These mechanisms may be involved in a complex cascade of cellular events associated with the pathophysiology of many types of cancer. MiRNAs are easily detectable in tissue and blood samples of cancer patients. Therefore, miRNAs hold good promise as potential biomarkers in ovarian cancer. In this review, we attempted to provide a comprehensive profile of key miRNAs involved in ovarian carcinoma to establish mi RNAs as more reliable non-invasive clinical biomarkers for early detection of ovarian cancer compared with protein and DNA biomarkers.

Expression of the novel gene NM23-H1B in ovarian cancer

Objective:To study the expression of the human novel gene NM23-H1B in ovarian cancer. Methods: Totally 24 samples from patients with epithelial ovarian tumor at different clinical stages and 4 from normal ovaries were examined for NM23-H1B mRNA expression by RT-PCR and Northern blot. Results: All samples expressed NM23-H1B mRNA through RT-PCR, while the level of expression in ovarian tumor was higher than that of normal ovary. The results of Northern blot showed that NM23-H1B was overexpressed in ovarian cancer while lowexpressed in normal ovary or low malignant potential (LMP). The level of expression at early stage cancer(stageⅠand Ⅱ) was higher than those in advanced cancer(stage Ⅲ and Ⅳ). In early stage carcinoma, the expression level was involved in the differentiation of tumor cell, and well-differentiated cancer expressed NM23-H1B mRNA in comparatively higher level. Conclusion: The novel gene NM23-N1B is closely correlated with the ovarian cancer.

The Application of HE4 in Diagnosis of Gynecological Pelvic Malignant Tumor

OBJECTIVE To investigate the value of human epididymis geneproduct 4 (HE4) in differential diagnosis of gynecological pelvictumors.METHODS The level of serum HE4 in 132 women wasdetermined. These women were divided into three groups, i.e.,46 women with good health being classified as the normal control(NC) group, and based on clinicopathological results, the other 86with pelvic masses being classified into groups of benign (n = 56)and malignant lesions (n = 30), respectively.RESULTS The range of serum HE4 in the NC group was(23.5～46.0) pmol/L, with an average value of (34.1 ± 5.6) pmol/L;the range of serum HE4 in the benign lesion group was (30.1～58.9)pmol/L, with an average value of (39.1 ± 7.2) pmol/L; the range ofserum HE4 in the group of malignancy was (31.2～1430.0) pmol/L,and the average value was (248.7 ± 364.5) pmol/L. The level ofHE4 in the malignant lesion group was significantly higher thanthat in the other 2 groups, with a statistical difference, P < 0.001.The diagnostic index reached maximum (0.847) when the serumHE4 was at 51.6 pmol/L, and the sensitivity and specificity of HE4were 86.7% and 98.0%, respectively. The area under the receiver-operator characteristic curve (ROC) was 0.935 (95% CI 0.832～1.037,P = 0.000). The consistency checking Kappa value of HE4 in thediagnosis of pelvic malignant tumors was 0.867, P = 0.000.CONCLUSION The determination of serum HE4 is a goodindicator in differential diagnosis of benign and malignant ovariantumors.

Reversing drug resistance in the ovarian carcinoma cell line SKOV3/mdr1 in vitro by antisense oligodeoxynucleotides

Objective To investigate the effect of multidrug resistance gene 1 (mdr1) antisense oligodeoxynucleotides (ODNs) on reversing multidrug resistance in the drug resistant ovarian carcinoma cell line SKOV3/mdr1. Methods The ovarian carcinoma cell line SKOV3 transducted with a human multidrug resistance gene (mdr1) served as the drug resistant model (SKOV3/mdr1). The mdr1 antisense ODNs was transfected into SKOV3/mdr1 cells while mediated by lipofectamine. Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the expression and the amount of the mdr1 mRNA in the cells. The positive rate and function of the mdr1 gene product P-glycoprotein (Pgp) in the mdr1 antisense ODNs treated SKOV3/mdr1 cells were determined by flow cytometry and rhodamine 123 efflux. Drug resistance in the SKOV3/mdr1 cell line was observed by MTT assay and cell colony culture. Results The mdr1 mRNA level was decreased to about 60% of that of β-actin after mdr1 antisense ODNs treatment. The Pgp positive rate of mdr1 antisense ODNs treated SKOV3/mdr1 cells decreased from 100% to 52.6% (P<0.01). The intracellular rhodamine 123 retention was increased from 9.1% to 33.8% (P<0.01). The chemoresistance to taxol decreased to 58% of SKOV3/mdr1 with mdr1 antisense ODN treatment. Compared with SKOV3/mdr1 cells in the control group, under a certain range of drug concentrations, the number of drug resistance colonies in mdr1 antisense ODNs treated SKOV3/mdr1 cells for taxol and doxorubicin decreased by 8.6±0.8 fold and 3.1±0.6 fold, respectively. Some non-specific functions during oligodeoxyncleotide treatment was also detected. Conclusion mdr1 expression in the SKOV1/mdr1 cell line was partially inhibited after mdr1 antisense ODNs treatment at the mRNA and protein level, increasing the chemotherapy sensitivity of this drug resistant ovarian carcinoma cell line.

Growth inhibition of interleukin-2 receptor gene-transduced peripheral blood lymphocytes on human ovarian cancer cells

Objective To investigate the growth inhibition of interleukin 2 receptor (IL 2R) gene transduced peripheral blood lymphocytes (PBLs) on human ovarian cancer cells Methods Interleukin 2 (IL 2) and IL 2R genes were transfected into human ovarian cancer cell line 3AO and PBLs, respectively, using the same Fugene vector Twenty four hours later transfected and nontransfected PBLs were cocultured with transfected and nontransfected 3AO for 48 hours Cytotoxity of PBLs on 3AO was detected by the MTT assay Results The morphology of IL 2 transduced 3AO and IL 2R transduced PBLs remained unchanged 3AO cells could be transfected with the IL 2 gene and expressed IL 2 mRNA, and PBLs could be transfected with the IL 2R gene and expressed IL 2R mRNA IL 2 transduced 3AO cells enhanced their response to the cytotoxity of PBLs Furthermore, growth inhibition of PBLs to 3AO cells increased significantly when the IL 2R was transfected into PBLs and when the IL 2 gene was transfected into 3AO cells and the two were combined Conclusions IL 2R gene transduced PBLs are able to enhance their cytotoxity on IL 2 gene transduced ovarian cancer cells This method may be a new way to investigate IL 2 gene therapy for ovarian cancer