沉默轴突导向蛋白4D基因对卵巢癌SKOV3细胞恶性生物学行为的影响

Objective To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude nice. Methods Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blotwere used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SK0V3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Results Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0. 401 ±0.051, 0. 120 ±0.035 and 0.014 ±0. 015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ±0.019, 0.536 ±0.040,respectively, P<0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0. 196 ± 0. 023, 0. 074 ± 0. 015 and 0. 040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0. 275 ± 0. 009, 0. 282 ± 0. 015, respectively, P < 0. 05 ). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR( inhibitory rate ) of pshRNASema4D-B group was ( 61.0 ± 3.3 ) % ( P < 0.05). Conclusions Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.     

沉默轴突导向蛋白4D基因对卵巢癌SKOV3细胞恶性生物学行为的影响

Objective To observe the effect of DNA Sema4D gene silencing by RNA interfering on the proliferation, migration and invasion of human ovarian cancer SKOV3 cells, and to study the effect of pshRNASema4D on the growth of SKOV3 cells in transplanted tumor in nude nice. Methods Recombinant plasmid pshRNASema4D-A, B and C were respectively transfected into SKOV3 cells by lipofetamine 2000, while cells transfected by plasmid vector pcDNA3.1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and Western blotwere used to detected the mRNA and protein expression of Sema4D in SKOV3 cells tranfected for 24, 48 and 72 hours. MTT assay was used to investigate the proliferation of the SKOV3 cells after trasnsfection. Transwell cell migration and invasion assays were used to investigate the migration and invasion abilities of the SK0V3 cells after trasnsfection. Human ovarian cancer model was established in nude mice, and the nude mice were treated with pshRNASema4D-B once every 3 days for 3 weeks. The bulk of the transplanted tumor was measured. Results Three Sema4D-targeted short hairpin RNA (shRNA) A, B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragment. The results indicated that both recombinant plasmid pshRNASema4D-A and B could effectively knock down the expression of Sema4D gene in human ovarian cancer cells, of which pshRNASema4D-B was the better choice, while no effect of pshRNASema4D-C was seen. RT-PCR results showed that the relative mRNA expression of Sema4D gene in SKOV3 cells transfected with pshRNA-Sema4D for 24, 48 and 72 hours were 0. 401 ±0.051, 0. 120 ±0.035 and 0.014 ±0. 015, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0.521 ±0.019, 0.536 ±0.040,respectively, P<0.05). The Westen blot analysis manifested that the relative expression of Sema4D protein of SKOV3 cells transfected by pshRNASema4D for 24, 48 and 72 hours were 0. 196 ± 0. 023, 0. 074 ± 0. 015 and 0. 040 ± 0.014, respectively, which were significantly lower than that in SKOV3 cells transfected by empty vector and non-transfected cells at 72 hours after transfection. (0. 275 ± 0. 009, 0. 282 ± 0. 015, respectively, P < 0. 05 ). Comparing with the empty vector-transfected and non-transfected cells, the proliferation, invasion and migration ability of SKOV3 cells transfected with pshRNA-Sema4D were obviously weakened. The pshRNASema4D-B significantly suppressed the growth of the SKOV3 cells-transplanted tumors in nude mice, and the IR( inhibitory rate ) of pshRNASema4D-B group was ( 61.0 ± 3.3 ) % ( P < 0.05). Conclusions Sema4D can be successfully silenced by RNA interfering in human ovarian cancer SKOV3 cells. Downregulation of Sema4D can inhibit the proliferation, migration and invasion of ovarian cancer cells. The pshRNASema4D can significantly suppress the growth in transplanted tumor of human ovary cancer in nude mice. Sema4D may become a candidate gene of gene therapy of human ovarian cancer.     

Effects of HLEC on the secreted proteins of epithelial ovarian cancer cells prone to metastasize to lymph nodes

Objective： To study explores the effect of HLEC on the secreted proteins of epithelial ovarian cancer （EOC） cells （SKOV3-PM4） with directional highly lymphatic metastasis. Methods： Supernatants of four groups of cultured cells, namely, SKOV3 （A）, SKOV3＋HLEC （B）, SKOV3-PM4 （C）, SKOV3-PM4＋HLEC （D）, were collected, and their proteins were detected by antibody arrays and iTRAOcZD-LC-MALDI- TOF/TOF/MS. Significantly differential proteins were further analyzed via bioinformatics and validated in human serums and cell media via ELISA. Results： Results of antibody arrays and mass spectrometry demonstrated that GRN and VEGFA were upregulated in group C （compared with group A）, whereas IGFBP7 and SPARC were downregulated in group D （compared with group C）. Comprehensive bioinformatics analysis results showed that IGFBP7 and VEGFA were closely linked to each other. Further validation with serums showed statistical significance in VEGFA and IGFBP7 levels among groups of patients with ovarian cancers, benign tumors, and control groups. Two proteins were upegulated in the first group. VEGFA in the control group was downregulated. For IGFBP, upregulation in the control group and down-regulation in the first group were also observed. Conclusion： The HLEC microenvironment is closely associated with directional metastasis to lymph nodes and with differential proteins including cell stromal proteins and adhesion factors. The upregulation of VEGFA and GRN and the downregulation of SPARC and IGFBP7 are closely associated with directional metastasis to lymph nodes in EOC cells.     

瘦素诱导乳腺癌细胞的凋亡抵抗及其分子机制

Objective To explore the apoptosis resistance induced by Leptin and its mechanism in breast cancer cells in vitro.Methods The leptin-mediated reduction of docetaxel-induced apoptosis in human breast cancer T47D cells was evaluated by TransAM ELISA,MTT and caspase-9 assay.The leptinpromoted survivin expression was analyzed by Western-blot and RT-PCR.The reversing effect of STAT3 knockdown on leptin-induced survivin upregulation was measured by Western-blot and RT-PCR.Results Leptin promoted T47D cells proliferation and the inhibitory rate was-63.6%.It reduced docetaxel-induced apoptosis in T47D cells by 31.9%.Leptin at different concentrations promoted survivin protein and mRNA expression in T47D cells.The expression of survivin mRNA was 4.6 fold compared with the T47D cells not treated with leptin(10 nmol/L).The expression of survivin mRNA in T47D cells was 0.55±0.15 fold after transfected with small interfering RNA(siRNA)of STAT3.The expression of survivin mRNA in STAT3 siRNA group and mock transfected group were 0.56±0.18 fold and 1.61±0.22 fold after treated by leptin,respectively.The survivin protein level of T47D mock transfected cells was increased after treated by leptin,but the protein level of T47D transfected with STAT3 siRNA cells were not changed significantly.Conclusion Leptin/STAT3 signaling is a novel pathway for up-regulation of survivin expression in breast cancer cells.     

Identification of differentially expressed long non-coding RNAs in human ovarian cancer cells with different metastatic potentials

Objective： To identify differentially expressed long non-coding RNAs （lncRNAs） involved in the metastasis of epithelial ovarian cancer. Methods： An in vitro invasion assay was performed to validate the invasive capability of SKOV3 and SKOV3.ip1 cell lines. Total R.NA was then extracted, and microarray analysis was performed. Moreover, nine lncRNAs were selected for validation using RT-qPCR. Results： Compared with the SKOV3 cells, the SKOV3.ip1 cells significantly improved in the in vitro invasive activity. Of the 4,956 lncRNAs detected in the microarra~ 583 and 578 lncRNAs were upregulated and downregulated, respectivel~ in SKOV3.ip1 cells, compared with the parental SKOV3 cells. Seven of the analyzed lncRNAs （MALAT1, H19, UCA1, CCAT1, LOC645249, LOC100128881, and LOC100292680） confirmed the deregulation found by microarray analysis. Conclusion： LncRNAs clusters were differentially expressed in ovarian cancer cells with varying metastatic potentials. This result indicates that some lncRNAs might exert a partial or key role in epithelial ovarian cancer metastasis. Further studies should be conducted to determine the roles of these lncRNAs in ovarian cancer metastasis.     

Retrovirus-Mediated CXCR4-siRNA Can Inhibit the Invasion of Prostate Carcinoma In Vitro

Objective： CXCR4 is a potential target for cancer gene therapy. In this study, an RNA interference retrovirus vector targeting CXCR4 gene was constructed, and its influence on the invasion of prostate cancer cells by depressing CXCR4 gene expression was analyzed. Methods： the CXCR4-specific siRNA gene was cloned by PCR and inserted into the Pgensil-1 plasmid eonstaining U6 promoter and EGFP, and then the recombinant fragment was sub-cloned into PLXSN and tested by restriction enzyme and sequencing. The virus obtained from transfected PA317 cells was transfected into prostate cancer cells. The expression of CXCR4 mRNA was detected by RT-PCR. The ability of invasion of prostate cancer cells in vitro was estimated by Transwell experiment. Results： it was showed that with the recombinant PLXSN transfection, the expression of CXCR4 mRNA in prostate cancer cells PC-3m and LNCaP was reduced at rates of （84.26±10.20）% and （88.17±11.23）%, respectively. The results of Transwell experiment also showed that the number of cells under micro-membrane were 14.7±3.1 and 18.9±4.2 in the treated group of PC-3m and LNCaP, respectively, compared with 46.9±5.3 and 66.7±6.0 in the control group （P〈0.05）. Conclusion： PLXSN/EGFP-U6-siCXCR4 can significantly depress the expression f CXCR4, by which the invasiveness of prostate cancer cells in vitro was inhibited as well. This recombinant fragment would be helpful in the treatment of prostate cancer.     

EFFECTS AND MECHANISMS OF ENDOSTATIN ON THE GROWTH OF OVARIAN CANCER SKOV3 CELLS IN VITRO AND IN VIVO

Objective： To evaluate the inhibitory effect of Endostatin on ovarian cancer cell line SKOV3 and to investigate the possible mechanism of the inhibition. Methods： Using MTT, transmission electron microscope （TEM） and immunocytochemistry, the effects of Endostatin on the proliferation of SKOV3 cells were studied. Nude mice were subcutaneously implanted with SKOV3 cells. The cell apoptosis of implanted tumor was detected by TUNEL and TEM. The expressions of bcl-2 and bax in implanted tumor tissues were measured by RT-PCR and immunohistochemistry. Results： Endostatin significantly inhibited the proliferation of SKOV3 cells in vitro （P〈0.01） and induced cell apoptosis, whereas the expressions of bcl-2 and bax were not changed obviously in SKOV3 cell treated with Endostatin. The mean tumor weight of Endostatin treated group was markedly lower than that of PBS control group （P〈0.05）. The expression of bcl-2 was down-regulated in Endostatin treated group, but bax was not influenced. Conclusions： The results demonstrated that Endostatin might have anti-tumor effect on ovarian carcinoma. One of the important mechanisms of Endostatin effect of anti-angiogenic and anti-tumor activities might involve regulating the bcl-2/bax expression and inducing apoptosis.     

MicroRNA-mRNA functional pairs for cisplatin resistance in ovarian cancer cells

Ovarian cancer is the leading cause of death in women worldwide. Cisplatin is the core of first-line chemotherapy for patients with advanced ovarian cancer. Many patients eventually become resistant to cisplatin, diminishing its therapeutic effect. MicroRNAs （miRNAs） have critical functions in diverse biological processes. Using miRNA profiling and polymerase chain reaction validation, we identified a panel of differentially expressed miRNAs and their potential targets in cisplatin-resistant SKOV3/DDP ovarian cancer cells relative to cisplatin-sensitive SKOV3 parental cells. More specifically, our results revealed significant changes in the expression of 13 of 663 miRNAs analyzed, including 11 that were up-regulated and 2 that were down-regulated in SKOV3/DDP cells with or without cisplatin treatment compared with SKOV3 cells with or without cisplatin treatment. miRNA array and mRNA array data were further analyzed using Ingenuity Pathway Analysis software. Bioinformatics analysis suggests that the genes ANKRD17, SMC1A, SUMO1, GTF2H1, and TP73, which are involved in DNA damage signaling pathways, are potential targets of miRNAs in promoting cisplatin resistance. This study highlights candidate miRNA-mRNA interactions that may contribute to cisplatin resistance in ovarian cancer.     

Down-Regulation of CXCR4 Expression by siRNA Inhibits Invasive Ability of Breast Cancer Cells

OBJECTIVE To investigate the efficiency of gene silencing by CXCR4- siRNAs (small interfering RNA), and to examine the invasive ability and the expression of other metastatic-associated genes in siRNA-treated breast cancer cells. METHODS Three siRNAs were designed and cloned into the pSilenc ? 3.1-H1 neo vector. The reconstructed plasmids were purified and trans- fected into the T47D breast cancer cell line, which highly expressed CXCR4. The amount of CXCR4 expression in the transfected cells was measured by flow cytometry and Real-time PCR. Cell invasive ability was evaluated us- ing 24-well Matrigel invasion chambers. In addition, the expression of other metastatic-associated genes, such as E-cad, IGFBP-5, FN and MMP-2, was assessed by Real-time PCR. RESULTS The suppression rates of CXCR4 mRNA expression reached 95.7%, 85.9% and 98.3%compared with control-siRNA cells in the 3 CXCR4- siRNA T47D cells respectively. FCM assays for CXCR4 protein expression showed a similar inhibitory effect. The invasion indexes of these CXCR4- siRNA cells were 0.037, 0.290 and 0.188 respectively compared with control- siRNA cells. After treatment of the cells with CXCR4-siRNA, the expression of E-cad showed an upward tendency and that of IGFBP-5 had a downward trend, while alteration in expression of FN and MMP2 varied without a con- sistant effect. CONCLUSION CXCR4 plays an important role in modulating migra- tion of human breast cancer cells. Small interfering RNA can significantly silence the CXCR4 gene in the human T47D breast cancer cell line. The results of this study strengthen the need for further research on novel gene therapy against breast cancer metastasis.     

siRNA 抑制Sema4C 基因表达对人乳腺癌细胞恶性行为的影响*

目的:研究小干扰RNA（small interf ering RNA,siRNA ）抑制轴突导向蛋白分子（Semaphorin 4C,Sema4C）基因的表达对人乳腺癌细胞MDA-MB-231 体外迁移、侵袭及增殖的影响,并初步探讨其作用机制。方法:根据Sema4C 基因设计序列特异性的siRNA（Sema4C-siRNA ）,在脂质体介导下转染MDA-MB-231 细胞,Western blot方法检测基因封闭效应,利用细胞划痕实验、Tran ?swell小室侵袭实验及CFSE流式细胞仪方法检测细胞转染前后迁移、侵袭及增殖能力的变化,Western blot检测磷酸化AKT（p-AKT ）在转染前后细胞中表达的变化。结果:转染Sema4C-siRNA 72小时后,乳腺癌MDA-MB-231 细胞株（MDA-MB-231/Si）Sema4C 蛋白表达明显下降,与未转染细胞MDA-MB-231 相比,MDA-MB-231/Si细胞体外迁移能力减弱,侵袭及增殖能力明显下降;p-AKT 表达水平在MDA-MB-231/Si细胞中明显降低。结论:Sema4C-SiRNA 转染人乳腺癌MDA-MB-231 细胞可下调细胞中Sema4C 蛋白表达水平,Sema4C-SiRNA 对MDA-MB-231 细胞的体外迁移、侵袭和增殖有抑制作用,这可能与p-AKT 的下调有关。      

人滋养层细胞表面抗原2的表达在卵巢癌侵袭转移中的作用及其机制

目的探讨人滋养层细胞表面抗原2(Trop2)在卵巢癌侵袭和转移中的作用及其分子机制。方法通过对Cancer Cell Line Encyclopedia(CCLE)和The Cancer Genome Atlas(TCGA)数据库进行数据挖掘,分析Trop2表达的临床意义。采用Western blot法检测卵巢癌细胞系A3O、A1780和SKOV3中Trop2蛋白的表达。采用Trop2-shRNA构建SKOV3-shRNA细胞模型,以定量逆转录聚合酶链反应检测SKOV3-shRNA和SKOV3-NC组细胞中SKOV3 mRNA的表达,以细胞计数盒8法检测SKOV3-shRNA组和SKOV3-NC组细胞的增殖,以流式细胞仪检测SKOV3-shRNA和SKOV3-NC组细胞的细胞周期和凋亡,以Transwell实验检测SKOV3-shRNA和SKOV3-NC组细胞的侵袭和迁移,以Western blot法检测SKOV3-shRNA和SKOV3-NC组细胞中AKT、p-AKT、β-catenin、caspase3、bcl-2、E-cadherin和vimentin蛋白的表达。结果 Trop2 mRNA在卵巢癌中高表达,其表达高低与肿瘤分期有关,也与患者预后有关。与A3O细胞比较,A1780和SKOV3细胞高表达Trop2蛋白(P<0.05)。SKOV3-NC组和SKOV3-shRNA组中Trop2 mRNA的相对表达量分别为1.18±0.24和0.42±0.08,差异有统计学意义(P<0.05)。SKOV3-NC组细胞的活力明显高于SKOV3-shRNA组(P<0.05)。SKOV3-NC和SKOV3-shRNA组G₀/G₁期细胞的比例分别为(38.67±4.22)%和(60.24±8.17)%,SKOV3-shRNA组细胞阻滞于G₀/G₁期(P<0.05)。SKOV3-shRNA组细胞的凋亡率为(26.32±1.81)%,明显高于SKOV3-NC组[(6.54±1.32)%,P<0.05]。SKOV3-shRNA组和SKOV3-NC组SKOV3细胞的迁移细胞数分别为(1 255.83±108.44)个/视野和(1 679.71±213.92)个/视野,侵袭细胞数分别为(242.49±52.09)个/视野和(473.54±73.11)个/视野,与SKOV3-NC组比较,SKOV3-shRNA组的迁移和侵袭细胞数均明显减少(均P<0.05)。SKOV3-shRNA细胞中p-AKT、bcl-2、vimentin、β-catenin表达下调,caspase3、E-cadherin表达上调,总AKT没有明显变化。结论 Trop2的表达与卵巢癌分期和预后有关,Trop2可通过激活AKT/β-catenin信号通路促进卵巢癌细胞增殖和转移,沉默Trop2的表达可以抑制卵巢癌进展。     

miR-101靶向甲基化转移酶3A抑制人卵巢癌细胞生长与侵袭

背景与目的：miR-101在胃癌、结肠癌、乳腺癌以及前列腺癌中表达下调,有类似抑癌基因样作用,然而,其在卵巢癌中的作用尚未明确。该研究旨在探讨miR-101是否通过靶向调控甲基化转移酶3A(DNMT3A)抑制人卵巢癌细胞生长与侵袭,从而进一步揭示miR-101的抑瘤机制。方法：采用实时定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)检测22例卵巢癌组织及癌旁正常卵巢组织中miR-101的表达改变;将miR-101 mimics转染于卵巢癌SKOV3细胞,以DNMT3A siRNA为阳性对照,采用蛋白［质］印迹法(Western blot)检测外源过表达miR-101对DNMT3A蛋白表达水平的影响;采用噻唑蓝(thiazolyl blue,MTT)和Transwell侵袭实验检测外源高表达miR-101对人卵巢癌细胞生长与侵袭能力的影响。结果：qRT-PCR检测结果显示,miR-101在22例卵巢癌组织中的表达水平较癌旁正常组织明显下调;Western blot检测结果显示,外源过表达miR-101或沉默DNMT3A能下调SKOV3细胞DNMT3A蛋白的表达水平;MTT检测结果显示,转染miR-101 mimics或沉默DNMT3A 48、72和96 h后D值与对照组比较明显减少,差异均有统计学意义(P<0.05);Transwell侵袭实验显示,转染miR-101 mimics或沉默DNMT3A 36 h后穿过基底膜的细胞数分别为(105±7)个和(107±13)个,与对照组(213±11)个比较能明显减缓SKOV3细胞的穿膜能力,差异有统计学意义(P<0.05)。结论：miR-101通过靶向调控DNMT3A抑制人卵巢癌细胞生长与侵袭。     

罗勒多糖对卵巢癌肿瘤转移相关基因SEMA4C表达影响实验研究

目的：观察分析罗勒多糖对卵巢癌SKOV3细胞肿瘤转移相关基因SEMA4C表达的影响,揭示罗勒多糖抗卵巢癌转移的作用机制。方法：体外培养卵巢癌SKOV3细胞,实验分为5组,A组为不加罗勒多糖处理的空白对照组,B组（罗勒多糖100mg／L）、C组（罗勒多糖500mg／L）、D组（罗勒多糖1000mg／L）、E组（紫杉醇0．05μmol／L）为紫杉醇组;荧光实时定量PCR检测不同处理组细胞中SEMA4CmRNA的表达变化;免疫细胞化学分析不同处理组细胞中se—ma4C蛋白的表达差异。结果：SKOV3细胞中SEMA4CmRNA相对表达量,B组为（3．60±0．01）×10-4,C组为（3．04±0．34）×10-4,D组为（1I81±0．42）×10-4,E组为（5．56±0．14）×10-4,与A组（9．02±0．41）×10-4比较,差异均有统计学意义,P〈0．01;B、C和D组与E组比较,差异有统计学意义,P〈0．01;SKOV3细胞中Sema4C蛋白表达平均光密度B组为9．64±0．21,C组为8．58±0．16,D组为6．15±0．38,E组为12．16±0．20,与A组的15．19±0．22比较,差异有统计学意义,P〈0．01;B、C和D组与E组比较,差异均有统计学意义,P〈O．01;罗勒多糖可以抑制卵巢癌细胞中SEMA4CmRNA及编码蛋白的表达,且随着药物浓度的增大,抑制作用越明显。结论：罗勒多糖可下调肿瘤转移相关基因SEMA4C的表达,可能是罗勒多糖抗卵巢癌淋巴道转移的新机制。     

外源性AKT1增强上皮性卵巢癌细胞侵袭和迁移能力的机制

探讨AKT1基因对上皮性卵巢癌SKOV3细胞迁移和侵袭能力的影响及相关分子机制。方法 针对AKT1基因,设计并构建shRNA质粒和真核表达质粒,双向调节SKOV3细胞中AKT1的表达,运用RT-PCR和western blot检测转染效率。运用Wound healing和Transwell-Matrigel方法检测转染前后细胞迁移和侵袭能力的变化。RT-PCR法检测与细胞运动侵袭相关分子CXCR4、VEGF、MMP-2、MMP-9和uPA在mRNA水平的表达变化。结果 成功构建AKT1基因的真核表达质粒pEF-1α-AKT1和靶向抑制AKT1基因的shRNA表达质粒pRNAT-AKT1。转染上皮性卵巢癌SKOV3细胞后,能有效调控p-AKT表达。参照未转染组和空载体转染组,外源性AKT1促进细胞迁移和侵袭,CXCR4、VEGF、MMP-2和uPA的mRNA表达水平升高。shRNA靶向抑制AKT1基因的表达可抑制细胞迁移和侵袭,CXCR4、VEGF、MMP-2和uPA的mRNA表达水平下降。结论 AKT1可能通过调控CXCR4、VEGF、MMP-2和uPA的转录水平来影响细胞侵袭和运动能力。     

SAHA对人卵巢癌SKOV3细胞转移潜能影响及其机制探讨

目的：观察组蛋白去乙酰化酶抑制剂辛二酰苯胺异羟肟酸（suberoylan．1idehydroxamicacid,SAHA）体外对人卵巢癌SKOV3细胞转移潜能的影响,并探讨其可能机制。方法：体外应用SAHA作用于人卵巢癌SKOV3细胞,实验分为对照组、4μmol／LSAHA组、8／μmol／LSAHA组和12μmol／LSAHA组,划痕实验检测SKOV3细胞迁移能力,Tran—swell侵袭实验检测SKOV3细胞侵袭能力。蛋白质印迹法检测SKOV3细胞内组蛋白H4乙酰化水平,实时定量PCR方法检测SKOV3细胞uPA和uPAR基因ITIRNA表达。结果：经sAHA作用48h后,对照组以及4、8和12μmol／L组细胞Ac—H4表达量分别为0．10±0．04、0．21±0．05、0．40±0．05和0．72±0．04,Ac—H4表达量随sAHA剂量浓度增加而增加,P〈0．001;各组细胞划痕愈合率分别为（68．27±2．98）0A、（35．19±2．97）％、（18．82±2．96）％和（10．24±2．98）％,划痕愈合率随sAHA剂量浓度增加而降低,P〈0．001;各组细胞穿过Transwell滤膜的细胞数量分别为58．89±4．35、37．66±4．27、21．15±4．32和10．75±4．30,穿过Transwell滤膜的细胞数量随sAHA剂量浓度增加而减少,P〈0．001;各组细胞uPArnRNA表达分别为22．48±1．05、15．40±1．02、8．62±1．05和5．274±1．08,随着sAHA剂量浓度增加而降低,P〈O．001;各组细胞uPAR基因mRNA表达量分别为18．22±12、12．37±1．14、7．64±1．10和3．55±1．12,随SAHA剂量浓度增加而降低,P〈0．001。结论：SAHA可抑制人卵巢癌SKOV3细胞迁移和侵袭能力,提高组蛋白乙酰化水平,降低uPA和uPAR基因表达可能是其作用机制。     

转化生长因子诱导的基因蛋白与肿瘤耐药的相关性

目的：探讨转化生长因子诱导的基因蛋白与肿瘤耐药的相关性。方法：分别用载有 TGFBI 基因质粒以及空质粒转染人卵巢癌细胞株 SKOV3,采用 Western blot 技术检测转染后两组细胞 TGFBI 蛋白的表达情况,用 MTT 法检测不同浓度紫杉醇、顺铂对上述两组细胞的抑制率,采用流式细胞技术检测紫杉醇对上述两组细胞凋亡的影响。结果：用载有 TGFBI 基因质粒转染细胞后其 TGFBI 蛋白表达情况显著高于用空质粒转染细胞后 TGFBI 蛋白的表达。在紫杉醇、顺铂同种药物浓度下,TGFBI 转染后细胞抑制率显著高于空质粒转染细胞（P <0.05）。TGFBI 质粒转染细胞凋亡率为（67.35±12.36）%,而空质粒转染细胞凋亡率为（50.21±14.85）%,TGFBI 质粒转染细胞凋亡率显著高于空质粒转染细胞凋亡率（P <0.05）。结论：TGFBI 高表达能够降低人卵巢癌细胞 SKOV3的耐药性,提高肿瘤细胞对化疗药物的敏感性,有助于促进肿瘤细胞的凋亡。     

siRNA干扰Id1表达增强卵巢癌细胞对顺铂的敏感性

[目的]探讨体外DNA结合抑制因子（inhibitor of DNA bindingor differentiation,Id）的表达对卵巢上皮癌SKOV3对顺铂化疗敏感性的影响。[方法]构建靶向Id1基因siRNA慢病毒载体并转染SKOV3细胞,筛选出稳定转染的SKOV3细胞克隆。实验分为4组：实验组（Cell＋LvshRNA-Id1）、阴性对照组（Cell＋Lv-shRNA-NC）、病毒对照组（Cell＋Lv-control）与空白对照组（Cell group）。CCK8检测各组SKOV3细胞不同时间增殖活性以及暴露于不同浓度顺铂（0.1、0.2、0.5、1、2、5、10μg/ml）48h后顺铂对各组SKOV3细胞的半数抑制浓度,分析沉默Id1基因后卵巢癌细胞对顺铂化疗敏感性的影响。[结果]三组对照组细胞增殖活性随着时间增加显著,而实验组则增加缓慢。在48h和72h时,实验组细胞增殖活性值分别为0.449±0.072μg/ml、0.885±0.232μg/ml,与三组对照组比较差异均有统计学意义（P均〈0.05）;实验组顺铂对SKOV3细胞的半数抑制浓度为1.5±0.71μg/ml,明显低于对照组（P〈0.01）。[结论]抑制Id1基因表达可以增加顺铂对卵巢癌细胞的生长抑制作用,为临床进一步提高卵巢癌的疗效提供了研究基础。     

DNMT1在卵巢癌组织中的表达及其对卵巢癌细胞增殖和迁移的影响

目的:探讨DNA甲基转移酶1(DNMT1)在卵巢癌组织中的表达及对卵巢癌细胞增殖和迁移能力的影响。方法:采用免疫组化方法检测15例正常卵巢组织及31例卵巢癌组织中DNMT1的表达水平;利用DNMT1的过表达质粒及DNMT1 siRNA瞬时转染SKOV3细胞系,Western blot检测DNMT1的蛋白表达水平,CCK8法检测各组细胞的增殖情况,Transwell小室实验检测DNMT1对卵巢癌细胞迁移能力的影响。结果:31例卵巢癌组织中DNMT1阳性率（83.9%,26/31）显著高于15例正常卵巢组织（20.0%,3/15）。临床分期Ⅲ-Ⅳ期、病理分级G3、远处转移者DNMT1阳性率明显升高（P＜0.05）。过表达DNMT1后,SKOV3细胞增殖能力及迁移能力均增强;敲低DNMT1后,SKOV3细胞生长及迁移能力受抑。结论:DNMTl在卵巢癌组织中的表达明显高于正常卵巢组织,其具有促进卵巢癌细胞增殖和远处转移的作用。     

下调视黄醇结合蛋白2基因表达对顺铂耐药卵巢癌细胞生物学特性的影响及其机制

目的探讨下调视黄醇结合蛋白2(RBP2)基因表达对卵巢癌细胞生物学特性的影响及其机制。方法建立稳定下调RBP2表达的顺铂(DDP)耐药卵巢癌细胞株SKOV3/DDP-RBP2i, 并设置阴性对照组和空白对照组。采用细胞计数盒8(CCK-8)法检测细胞增殖能力, 流式细胞术检测细胞凋亡情况, 划痕实验和Transwell侵袭实验检测细胞迁移和侵袭能力, 实时荧光定量聚合酶链反应(RT-qPCR)和Western blot法检测上皮-间质转化(EMT)相关分子标志物的表达。通过裸鼠移植瘤实验验证RBP2对卵巢癌生长的影响, 采用美国TCGA数据库中的卵巢癌临床数据分析RBP2表达与肿瘤转移和患者预后的关系。结果下调RBP2的表达后, SKOV3/DDP细胞的增殖能力明显下降, 第5天时, SKOV3/DDP-RBP2i组、阴性对照组和空白对照组的增殖活性分别为(56.67±4.16)%、(84.67±3.51)%和(87.00±4.00)%, 差异开始有统计学意义(P<0.001)。SKOV3/DDP-RBP2i组的凋亡率为(14.19±1.50)%, 高于阴性对照组[(8.77±0...