内源性IL-8诱导卵巢癌细胞对顺铂与紫杉醇产生耐药的机制研究

目的探讨内源性IL-8诱导卵巢癌细胞对顺铂和紫杉醇产生耐药的机制及相关信号转导通路。方法在原有工作基础上,以2种人卵巢癌细胞系A2780(不分泌IL-8,对顺铂、紫杉醇敏感)和SKOV-3(高分泌IL-8,对顺铂、紫杉醇耐药)为研究模型,分别将正义(sense,ss)IL-8基因或反义(antisense,as)IL-8基因稳定转染至A2780细胞或SKOV3细胞,应用MTT法、Caspase-3活性测定、RT-PCR及Western blot技术等观察内源性IL-8是否影响卵巢癌细胞对顺铂和紫杉醇的敏感性,并对其作用的机制和可能的信号传导通路进行研究。结果 1)内源性过表达IL-8可诱导A2780细胞对顺铂和紫杉醇产生耐药,而抑制IL-8表达可恢复SKOV3细胞对顺铂和紫杉醇的敏感性,IL-8诱导的卵巢癌细胞化疗耐药是通过降低Caspase-3活性来实现的;2)内源性过表达IL-8可上调A2780细胞的耐药相关基因MDR1和凋亡抑制基因Bcl-2、Bcl-xL及XIAP的表达,而抑制IL-8表达可使上述基因的表达明显降低;3)Wortmannin(PI3K抑制剂)和PD98059(MEK1/2抑制剂)能分别阻断IL-8诱导下卵巢癌细胞的Akt和ERK活化及化疗耐药作用。结论 IL-8诱导的卵巢癌细胞化疗耐药可能与其上调耐药相关基因MDR1和凋亡抑制基因Bcl-2、Bcl-xL及XIAP的表达以及活化Raf/MEK/ERK和PI3K/Akt信号通路相关,提示调节IL-8表达或其相关信号通路可能是治疗耐药性卵巢癌的一种良好策略。     

人上皮性卵巢癌细胞系侧群细胞的肿瘤干细胞样细胞生物学特性及膜蛋白差异表达的鉴定

目的 鉴定上皮性卵巢癌细胞侧群细胞(SP细胞)肿瘤干细胞样细胞生物学特性,并检测侧群细胞内差异蛋白质的表达.方法 流式分选上皮性卵巢癌细胞系SKOV3和A2780具有外泌Hochest33342染料生物学特性的侧群细胞,鉴定其肿瘤干细胞样细胞相关生物学特性.运用细胞培养稳定同位素标记(SILAC)的定量蛋白质组学技术,...     

LncRNA MEG3 impacts proliferation,invasion, and migration of ovarian cancer cells through regulating PTEN

Objective and design

We investigated the expressions of lncRNA MEG3 and PTEN in ovarian cancer tissues and their effects on cell proliferation, cycle and apoptosis of ovarian cancer.

Methods

Expression levels of MEG3 in ovarian cancer cell lines and normal ovarian cell lines were detected by qRT-PCR. Cell viability was detected by MTT assay. Cell apoptosis and cell cycle distribution were measured by flow cytometry. Cell invasion capability was tested by transwell assay. Cell migration capacity was tested by wound healing. The xenograft model was constructed to explore the effect of lncRNA MEG3 on ovarian cancer in vivo.

Result

Compared with normal ovarian cells, expression levels of MEG3 and PTEN were relatively lower in ovarian cancer cells. There was a positive correlation between the expression of PTEN and the expression of MEG3. Enhanced expression level of PTEN suppressed SKOV3 cell proliferation, increased cell apoptosis rate, and decreased cell invasion and migration.

Conclusion

LncRNA MEG3 and PTEN were down-regulated in ovarian cancer cells. LncRNA MEG3 regulated the downstream gene PTEN in ovarian cancer cells to prohibit cell proliferation, promote apoptosis and block cell cycle progression.

     

靶向上皮性卵巢癌SKOV3细胞ZEB1-shRNA重组体构建及功能研究

目的:利用RNA干扰技术,降低人上皮性卵巢癌SKOV3细胞中锌指增强子结合蛋白1(ZEB1)基因的表达,观察ZEB1低表达对SKOV3细胞的生长影响。方法:用pSUPER-EGFP1载体构建针对人ZEB1基因的干扰质粒shZEB1,脂质体转染SKOV3细胞,G418筛选稳定转染细胞株,通过RT-PCR和Western blot检测ZEB1在SKOV3细胞中表达。用克隆形成、划痕试验、RT-PCR和致瘤试验分别检测转染细胞克隆能力、迁移力、miR-200c表达水平和在裸鼠的致瘤性。结果:成功构建shZEB1,稳定转染细胞株shZEB1-SKOV3内ZEB1表达下降,导致shZEB1-SKOV3细胞miR-200c表达增高,克隆能力、迁移力及致瘤性下降。结论:用RNA干扰技术可靶向降低卵巢癌细胞株SKOV3内ZEB1的表达,使其致瘤性降低,提示ZEB1可作为分子靶点用于上皮性卵巢癌靶向治疗。     

miR-194-5p inhibits SLC40A1 expression to induce cisplatin resistance in ovarian cancer

BackgroundmiR-194-5p has been associated with drug resistance in many cancers. However, the role of miR-194-5p in cisplatin resistance in ovarian cancer is still unclear.Materials and methodsTo study the role and mechanism of miR-194-5p in cisplatin resistance, qRT-PCR was performed to determine the expression of miR-194-5p and SLC40A1 in ovarian cancer. Cell Counting Kit-8 (CCK8) assay was used to analyse cell viability after cisplatin treatment. Dual-luciferase reporter gene assay was performed to examine the relationship between miR-194-5p and SLC40A1. The genes downstream of SLC40A1 were investigated through bioinformatics analysis.ResultsCompared to cisplatin-sensitive ovarian cancer cells, higher miR-194-5p expression and lower SLC40A1 expression were found in cisplatin-resistant ovarian cancer cells. Moreover, this study demonstrated that over-expression of miR-194-5p inhibited SLC40A1 expression, and knockdown of miR-194-5p promoted SLC40A1 expression. In addition, dual-luciferase reporter gene assay further confirmed the negative correlation between miR-194-5p and SLC40A1. Furthermore, we found that over-expression of miR-194-5p resulted in cisplatin resistance. When miR-194-5p and SLC40A1 were simultaneously up-regulated, cisplatin sensitivity increased, while down-regulation of miR-194-5p sensitised ovarian cancer cells to cisplatin. However, when miR-194-5p and SLC40A1 were simultaneously down-regulated, cisplatin sensitivity was decreased. These data suggested that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance. In addition, bioinformatics analysis indicated a positive correlation of SLC40A1 with hephaestin (HEPH), and homeostatic iron regulator (HFE). However, we found that HEPH and HFE were associated with cisplatin resistance, suggesting that their role in drug resistance is induced by miR-194-5p/SLC40A1.ConclusionIn conclusion, we found that miR-194-5p inhibited SLC40A1 expression to induce cisplatin resistance in ovarian cancer. This study suggests that miR-194-5p could be a potential therapeutic target and a prognostic biomarker for ovarian cancer, with important implications for future research.     

JAK2,HSP基因蛋白表达与卵巢癌紫杉醇化疗耐药相关性的研究

目的检测卵巢癌细胞株及紫杉醇化疗后卵巢癌组织中JAK2,HSP,基因蛋白表达的变化,探讨其与卵巢癌紫杉醇化疗耐药的关系及临床意义。方法选用卵巢癌紫杉醇耐药细胞株SKOV3/TAX及敏感细胞株SKOV3,及58例卵巢癌组织,包括卵巢癌术后及紫杉醇化疗后复发患者19例（化疗组）,术前未化疗患者39例（未化疗组）。采用免疫组化二步法,检测卵巢癌组织及细胞中JAK2,HSP基因的蛋白表达。结果 1.光镜下观察SKOV3/TAX细胞株中JAK2、HSP蛋白表达强度均明显高于SKOV3细胞株;组织中化疗组与未化疗组相比JAK2,HSP的蛋白表达累积光密度值均明显增加（分别为：36987.38±16873.42 vs 27756.29±7136.09;34905.64±12361.87 vs 26684.56±12662.24）,差异均有统计学意义（P〈0.05）。2.各基因蛋白表达在临床分期,分化程度和病理类型中均无显著性差异。3.JAK2、HSP基因蛋白表达两者间呈正相关关系（r=0.330,P=0.011）。结论 JAK2、HSP基因蛋白表达上调可能与卵巢癌紫杉醇化疗耐药有一定相关性。     

MicroRNA-101 inhibits growth and metastasis of human ovarian cancer cells by targeting PI3K/AKT

IntroductionOvarian cancer is the most frequent cause of gynecological cancer related mortality in woman. This study was designed to investigate the role and therapeutic potential of miRNA-101 in ovarian cancer.Material and methodsExpression analysis was carried out by real-time quantitative polymerase chain reaction. Transfections were performed with the help of Lipofectamine 2000 reagent. AO/EB and annexin V/PI staining was used to detect apoptosis and flow cytometry was used for cell cycle analysis. Western blotting was employed for cell cycle analysis.ResultsIt was found that miRNA-101 was significantly down-regulated in ovarian cancer cells. The over-expression of miRNA-101 causes a significant decrease in the viability of ovarian cancer cells via the initiation of apoptosis and sub-G1 arrest of OVACAR-3 cells. It was indicated that PTEN was the potential target of miRNA-101 in OVACAR-3 cells. There was 4.5-fold up-regulation of PTEN expression in ovarian cancer cell lines and the over-expression of miRNA-101 in OVACAR-3 cells resulted in the down-regulation of PTEN expression. The inhibition of PTEN in the OVACAR-3 cells arrested the proliferation of these cells. The over-expression of miRNA-101 causes significant down-regulation in PI3K and AKT expression of OVACAR-3 cells.ConclusionsIt can be concluded that miRNA-101 acts as a tumor suppressor which may be beneficial in the treatment of ovarian cancer.     

Analysis of microarray-identified genes and microRNAs associated with drug resistance in ovarian cancer

The aim of this study was to identify potential microRNAs and genes associated with drug resistance in ovarian cancer through web-available microarrays. The drug resistant-related microRNA microarray dataset GS54665 and mRNA dataset {"type":"entrez-geo","attrs":{"text":"GSE33482","term_id":"33482"}}GSE33482, {"type":"entrez-geo","attrs":{"text":"GSE28646","term_id":"28646"}}GSE28646, and {"type":"entrez-geo","attrs":{"text":"GSE15372","term_id":"15372"}}GSE15372 were downloaded from the Gene Expression Omnibus database. Dysregulated microRNAs/genes were screened with GEO2R and were further identified in SKOV3 (SKOV3/DDP) and A2780 (A2780/DDP) cells by real-time quantitative PCR (qRT-PCR), and then their associations with drug resistance was analyzed by comprehensive bioinformatic analyses. Nine microRNAs (microRNA-199a-5p, microRNA-199a-3p, microRNA-199b-3p, microRNA-215, microRNA-335, microRNA-18b, microRNA-363, microRNA-645 and microRNA-141) and 38 genes were identified to be differentially expressed in drug-resistant ovarian cancer cells, with seven genes (NHSL1, EPHA3, USP51, ZSCAN4, EPHA7, SNCA and PI15) exhibited exactly the same expression trends in all three microarrays. Biological process annotation and pathway enrichment analysis of the 9 microRNAs and 38 genes identified several drug resistant-related signaling pathways, and the microRNA-mRNA interaction revealed the existence of a targeted regulatory relationship between the 9 microRNAs and most of the 38 genes. The expression of 9 microRNAs and the 7 genes by qRT-PCR in SKOV3/DDP and A2780/DDP cells indicating a consistent expression profile with the microarrays. Among those, the expression of EPHA7 and PI15 were negatively correlated with that of microRNA-141, and they were also identified as potential targets of this microRNA via microRNA-mRNA interaction. We thus concluded that microRNA-141, EPHA7, and PI15 might jointly participate in the regulation of drug resistance in ovarian cancer and serve as potential targets in targeted therapies.     

外源性IL-8诱导卵巢癌细胞对顺铂和紫杉醇产生耐药的机制及相关信号转导通路的初步探讨

目的探讨外源性IL-8诱导卵巢癌细胞对顺铂和紫杉醇产生耐药的机制及相关信号转导通路。方法在原工作基础上,以两种人卵巢癌细胞系A2780(不分泌IL-8,对顺铂、紫杉醇敏感)和SKOV-3(高分泌IL-8,对顺铂、紫杉醇耐药)为研究模型,观察外源性IL-8是否影响卵巢癌细胞对顺铂和紫杉醇的敏感性,并对其作用的机制和可能的信号转导通路进行研究。结果①体外经IL-8处理的A2780细胞对顺铂和紫杉醇的敏感性均明显降低即产生部分耐药(耐药倍数分别为6.07和7.23),其耐药程度均低于IL-8高表达的SKOV-3细胞(耐药倍数分别为8.22和9.03)。②IL-8可以剂量依赖的方式上调A2780和SKOV-3细胞的耐药相关基因MDR1和凋亡抑制基因Bcl-2、Bcl-xL及XIAP的表达。③MEK1/2抑制剂和PI3K抑制剂均能阻断IL-8诱导的卵巢癌细胞对顺铂和紫杉醇产生的耐药。结论IL-8-很可能通过上调耐药相关基因MDR1和凋亡抑制基因Bcl-2、Bcl-xL及XIAP的表达而致卵巢癌细胞对顺铂和紫杉醇产生耐药。IL-8的上述作用是由Ras/Raf/MEK/MAPK和PI3K/Akt信号通路介导的。     

CD44和NANOG在上皮性卵巢癌中的表达及其临床意义

目的:探讨上皮性卵巢癌(epithelial ovarian cancer,EOC)中CD44及转录因子NANOG的表达及其临床意义。方法:用免疫组织化学法检测良性卵巢肿瘤和EOC组织中CD44及NANOG的表达,并对EOC中CD44和NANOG的表达水平做相关性分析。Western blot法检测不同浓度顺铂对卵巢癌SKOV3细胞中CD44及NANOG蛋白表达水平的影响。结果:CD44和NANOG在EOC中的阳性表达率均明显高于良性卵巢肿瘤组织(P0.01);EOC中CD44与NANOG的表达水平呈正相关(r=0.346,P0.01)。CD44在EOC中的阳性表达率与临床分期和淋巴结转移有关(P0.05),而与年龄、病理分级、病理类型和肿瘤位置无关;NANOG在EOC中的阳性表达率与病理分级和临床分期有关(P0.05),而与年龄、病理类型、淋巴结转移和肿瘤位置无关。顺铂可诱导卵巢癌SKOV3细胞中CD44和NANOG表达上调(P0.05)。结论:CD44和NANOG在EOC中高表达,且与EOC发生和发展有关。顺铂可诱导卵巢癌SKOV3细胞中肿瘤干细胞标记物CD44及胚胎干细胞多能性基因产物NANOG表达升高。肿瘤干细胞的产生可能是促进EOC发生、发展及化疗耐药的潜在机制。     

慢病毒载体抑制FOXM1对卵巢癌SKOV3细胞增殖的影响与分子机制

目的:研究靶向转录因子FOXM1 shRNA慢病毒载体抑制FOXM1表达对人卵巢癌SKOV3细胞增殖的影响及其分子机制。方法:应用感染复数(MOI)为20的靶向FOXM1shRNA慢病毒颗粒感染人卵巢癌SKOV3细胞,MTT比色法检测感染后SKOV3细胞的生长曲线;流式细胞术分析感染72 h后细胞周期变化;实时荧光定量PCR及Western blot法分别检测感染72 h后FOXM1、Cyclin D1、PLK1等基因mRNA及蛋白表达变化。结果:MOI为20的靶向FOXM1 shRNA慢病毒颗粒能显著抑制SKOV3细胞生长,并将细胞阻滞在G0/G1期而S期细胞减少(P<0.01);显著下调FOXM1、CyclinD1、PLK1等基因mRNA及蛋白表达。结论:抑制FOXM1表达对人卵巢癌SKOV3细胞具有显著的增殖抑制作用。其作用与下调Cyclin D1、PLK1等蛋白的表达将细胞阻滞在G0/G1期有关。     

NEK2基因沉默促进卵巢癌细胞SKOV3凋亡

目的研究siRNA干扰NEK2表达对卵巢癌细胞SKOV3增殖和凋亡的影响及其分子机制。方法设计合成3条对NEK2的siRNA,转染进SKOV3细胞,采用real-time PCR和Western blot技术筛选出抑制效率最高的NEK2-siRNA序列,进行后续实验;分别采用MTT和流式细胞学技术检测细胞增殖和凋亡,用Western blot检测BAD、BCL-2和caspase-3的表达。结果 1)RNA干扰序列2对SKOV3细胞NEK2的抑制效果最明显;2)与空白组和阴性对照组相比,NEK2-siRNA转染48 h后,SKOV3细胞增殖能力下降,发生凋亡的细胞增加(P<0.01);3)与空白组和阴性对照组相比,NEK2-siRNA转染48 h后,SKOV3细胞中BAD和caspase-3的蛋白表达上调,而BCL-2的蛋白表达下调(P<0.01)。结论 NEK2-siRNA可能通过沉默NEK2的表达,促进卵巢癌细胞SKVO3的凋亡。     

MCT1 promotes the cisplatin-resistance by antagonizing Fas in epithelial ovarian cancer

This study was designed to investigate the role of MCT1 in the development of cisplatin-resistant ovarian cancer and its possible relationship with Fas. We found the expression of MCT1 was obviously increased both in cisplatin-resistant ovarian cancer tissue and A2780/CP cells compared with sensitive ovarian cancer tissue and cell lines A2780. And in A2780 cells treated with Cisplatin, the expression of MCT1 increased in a concentration-dependent manner, MCT1 knockdown attenuates cisplatin-induced cell viability. In A2780 and A2780/CP cells transfected with MCT1 siRNA, the activation of several downstream targets of Fas, including FasL and FAP-1 were largely prevented, whereas the expression of Caspase-3 was increased, accompanying with increased abundance of Fas. Coimmunoprecipitation and immunofluorescence showed that there is interaction between endogenous MCT1 with Fas in vivo and in vitro. In vivo, depletion of MCT1 by shRNA reverses cisplatin-resistance and the expression of Fas. This study showed that down regulation of MCT1 promote the sensibility to Cisplatin in ovarian cancer cell line. And this effect appeared to be mediated via antagonizing the effect of Fas.     

miR-141-3p对卵巢癌的作用及其机制

目的:探究miR-141-3p在卵巢癌中的作用及其相关的分子机制.方法:实时荧光定量PCR检测30例卵巢囊肿和30例卵巢癌组织中miR-141-3p和表皮生长因子受体(EGFR)的表达水平.将SKOV3细胞分为NC组(无转染的SKOV3细胞),miR-141-3p组(SKOV3细胞转染miR-141-3p),LV-EG...     

Systemic mesenchymal stem cells reduce growth rate of cisplatin-resistant ovarian cancer

Epithelial ovarian cancer is one of the most malignant cancers in women and resistant to chemotherapy is the major obstacle for the five-year survival rate. Cisplatin is one of the effective anticancer drug used in the ovarian cancer. To find a good strategy to cure the tumors which is resistant to cisplatin, the cisplatin-resistant 3SKOV3 cells were selected from SKOV-3 ovarian cancer cells. Furthermore, the isolated mesenchymal stem cells were infused systemically to try to cure the transplanted tumor induced by 3SKOV3 cells in nude mice. The morphology and cell membrane CD44 expression were investigated by microscope and flow cytometry. The biological behaviors of resistant 3SKOV3 and its parental SKOV3 cells, including proliferation, adhesion, and cell cycle were determined by CCK8, absorbance assay and FCM methods. The transplanted tumors were set up in nude mice with 3SKOV3 cells injection. The growth rate of transplanted tumors was detected following with MSCs injection. The 3SKOV3 cells have different morphologic manifestation and expressed high level of CD44 molecule. At the same time, 3SKOV3 cells have less adhesion ability and less S-phase ratio. The isolated MSCs from bone marrow could inhibit the growth of transplanted tumor via systemic injection. The cisplatin-resistant 3SKOV3 cells have the different biological behaviors as its parental SKOV3 cells. The present study indicated that systemic MSCs have the therapeutic role on ovarian cancer. However, further investigations are in progress to elucidate the underlying mechanism.     

过表达细胞黏附分子1抑制卵巢癌细胞迁移及侵袭

目的:研究过表达细胞黏附分子1(cell adhesion molecule 1,CADM1)对卵巢癌增殖、迁移和侵袭的影响.方法:qRT-PCR测定CADM1mRNA在卵巢癌细胞系SKOV3及人正常卵巢上皮细胞hose中的表达,将SKOV3细胞分成两组,即CADM1过表达组和对照组,转染48 h后Western印迹测定两组CADM1蛋白表达量,采用lipofectamine 2000分别转染pcDNA3.1-CADM1及pcDNA3.1质粒,采用CCK-8、克隆形成、细胞划痕及Transwell实验分别检测两组细胞增殖、克隆形成、细胞迁移及侵袭能力.结果:CADM1mRNA在SKOV3中表达水平显著低于hose细胞系(1.54±0.34 vs.5.63±0.96,p＜0.05);转染48 h后,CADM1过表达组和对照组CADM1蛋白表达量分别为2.53±0.42,0.37±0.09,差异具有统计学意义(P＜0.05).CADM1过表达组和对照组在0,24,48,72 h 450 nm处的OD值差异无统计学意义(P＞0.05);CADM1过表达组与对照组克隆形成数相比(60.4±7.6 vs.58.3±8.2),差异无统计学意义(P＞0.05);CADM1过表达组细胞迁移率显著低于对照组(20.3％±3.5％vs.60.1％±4.2％,P＜0.05);CADM1过表达组侵袭细胞数显著少于对照组(24.5±5.3 vs.65.1±6.9,P＜0.05).结论:CADM1在卵巢癌细胞系中低表达,过表达CADM1对卵巢癌细胞增殖和克隆形成无影响,但可抑制迁移和侵袭,起抑癌基因的作用.     

Low MAD2 expression levels associate with reduced progression-free survival in patients with high-grade serous epithelial ovarian cancer

Epithelial ovarian cancer (EOC) has an innate susceptibility to become chemoresistant. Up to 30% of patients do not respond to conventional chemotherapy [paclitaxel (Taxol^®) in combination with carboplatin] and, of those who have an initial response, many patients relapse. Therefore, an understanding of the molecular mechanisms that regulate cellular chemotherapeutic responses in EOC cells has the potential to impact significantly on patient outcome. The mitotic arrest deficiency protein 2 (MAD2), is a centrally important mediator of the cellular response to paclitaxel. MAD2 immunohistochemical analysis was performed on 82 high‐grade serous EOC samples. A multivariate Cox regression analysis of nuclear MAD2 IHC intensity adjusting for stage, tumour grade and optimum surgical debulking revealed that low MAD2 IHC staining intensity was significantly associated with reduced progression‐free survival (PFS) (p = 0.0003), with a hazard ratio of 4.689. The in vitro analyses of five ovarian cancer cell lines demonstrated that cells with low MAD2 expression were less sensitive to paclitaxel. Furthermore, paclitaxel‐induced activation of the spindle assembly checkpoint (SAC) and apoptotic cell death was abrogated in cells transfected with MAD2 siRNA. In silico analysis identified a miR‐433 binding domain in the MAD2 3′ UTR, which was verified in a series of experiments. Firstly, MAD2 protein expression levels were down‐regulated in pre‐miR‐433 transfected A2780 cells. Secondly, pre‐miR‐433 suppressed the activity of a reporter construct containing the 3′‐UTR of MAD2. Thirdly, blocking miR‐433 binding to the MAD2 3′ UTR protected MAD2 from miR‐433 induced protein down‐regulation. Importantly, reduced MAD2 protein expression in pre‐miR‐433‐transfected A2780 cells rendered these cells less sensitive to paclitaxel. In conclusion, loss of MAD2 protein expression results in increased resistance to paclitaxel in EOC cells. Measuring MAD2 IHC staining intensity may predict paclitaxel responses in women presenting with high‐grade serous EOC. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

FTO accelerates ovarian cancer cell growth by promoting proliferation,inhibiting apoptosis,and activating autophagy

ObjectiveFat mass and obesity-associated protein (FTO) is identified as a critical demethylase involved in various physiological processes. Despite efforts have been made to study the biological functions of FTO in certain cancers, the role of FTO in ovarian cancer is largely unknown. In this study, we sought to investigate the function of FTO on proliferation, apoptosis and autophagy of ovarian cancer cells.MethodsQuantitative real-time PCR was performed to detect FTO expression in ovarian tumor tissues and ovarian cancer cell lines OVCAR-3, SKOV-3, COC1, HO-8910 and A2780. SKOV-3 cells were constructed with FTO overexpression and A2780 cells were constructed with FTO knockdown. CCK-8 assay was used to examine cell viability and flow cytometry was used to detect cell apoptosis. Activity assay kits were applied to detect caspase-3 and caspase-9 levels. Western blot was performed to measure the expressions of FTO, PCNA, Bax, Bcl-2, LC3, ATG5, P62, p-AKT and AKT. Stable FTO-overexpression SKOV-3 cells or FTO-depletion A2780 cells were injected subcutaneously into male Balb/c-nu mice. Xenografted tumors were assayed by H&E staining. Immunohistochemistry was subjected to measure FTO and Ki67 expressions.ResultsFTO was up-regulated in ovarian tumor tissues compared with non-cancerous ovarian tissues. FTO overexpression markedly increased viability and autophagy function, but decreased apoptosis of ovarian cancer cells. In addition, FTO overexpression promoted AKT phosphorylation. In contrast, FTO silence showed the opposite effect.ConclusionFTO accelerated ovarian cancer cell growth by promoting proliferation, inhibiting apoptosis, and activating autophagy.     

高表达IL-12卵巢癌细胞的建立

目的 建立高表达IL-12的卵巢癌细胞,为后续IL-12治疗卵巢癌奠定基础。方法 将人卵巢癌细胞株SKOV3分为SKOV3组、SKOV3/GFP组和SKOV3/IL-12组三个实验组,SKOV3组不做任何处理,SKOV3/GFP组给予空载病毒感染SKOV3细胞,SKOV3/IL-12组给予IL-12基因腺病毒感染SKOV3细胞。腺病毒感染SKOV3细胞48 h后,使用荧光显微镜检测三个实验组的感染效率,RT-PCR检测各组SKOV3细胞IL-12 p35、p40 mRNA水平的表达量,ELISA方法检测感染72 h后各组细胞培养液中IL-12的含量。结果 SKOV3/GFP和SKOV3/IL-12组中的SKOV3细胞均可以在荧光显微镜下观察到绿色荧光,而SKOV3组没有观察到绿色荧光,SKOV3/GFP和SKOV3/IL-12组的感染效率分别是（97±1）%和（95±1）%,差异具有统计学意义（P＜0.05）;RT-PCR结果表明,与SKOV3和SKOV3/GFP组相比较,SKOV3/IL-12组可以成功扩增出IL-12 p35、p40亚基,差异具有统计学意义（P＜0.05）;ELISA结果表明,与SKOV3和SKOV3/GFP组比较,SKOV3/IL-12组能够高表达IL-12P70蛋白,差异具有统计学意义（P＜0.05）。结论 成功建立了高表达IL-12的卵巢癌SKOV3细胞,为进一步采用IL-12进行卵巢癌的免疫治疗打下了基础。     

Fibulin-3表达对恶性胸膜间皮瘤细胞的影响

目的:研究纤蛋白3(fibulin-3)基因过表达/沉默对人恶性胸膜间皮瘤(malignant pleural mesothelioma,MPM)细胞系SMC-1的影响,为MPM的治疗寻找新的方法。方法:分别构建fibulin-3过表达和短发夹RNA(short hairpin RNA,shRNA)载体,并将SMC-1细胞分别转染空白对照载体(control)、过表达载体(Exp)、过表达对照载体(Exp-NC)、干扰载体(shRNA1、shRNA2、shRNA3和shRNA4)和干扰对照载体(shRNA-NC)。应用流式细胞术检测细胞周期及凋亡,real-time PCR法和Western blot法检测过表达/干扰前后fibulin-3的表达情况。结果:SMC-1细胞转染相应载体后,流式细胞术结果显示,与control组相比,fibulin-3+Exp组的G_2期细胞数量增加(P0.05),shRNA2组的G_2期数量减少(P0.05);凋亡检测结果显示,与control组相比,Exp组的细胞凋亡率下降(P0.05),而shRNA2组的细胞凋亡率上升(P0.05)。分子水平检测结果显示,与control组相比,Exp组fibulin-3和间皮素(mesothelin)的mRNA和蛋白表达水平上调(P0.05),各shRNA组fibulin-3和mesothelin的mRNA和蛋白表达水平下调(P0.05)。结论:本实验构建了fibulin-3基因的过表达及沉默载体,证实转染Exp和shRNA能有效改变fibulin-3的表达水平及SMC-1细胞的生长,为以fibulin-3为靶点的RNA沉默手段应用于临床MPM的治疗提供了实验依据。