Ⅰ型与Ⅱ型子宫内膜癌差异表达microRNA的筛选及其靶基因的研究
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摘要
目的筛选Ⅰ型和Ⅱ型人子宫内膜癌细胞系中差异表达的miRNA并预测和初步验证其靶基因。
方法(1)通过体内成瘤病理学检查、免疫组化检测ER、PR、p53和Ki-67的表达、对雌孕激素作用的反应来鉴定两个细胞系Ishikawa和KLE的分型;(2)无雌孕激素环境下培养细胞系Ishikawa和KLE,提取总RNA进行miRNA芯片检测和基因表达谱芯片检测;提取总蛋白进行蛋白质组学分析;(3)用miRANDA和TargetScan软件结合芯片和蛋白质分析结果,预测差异表达的miRNA可能的靶基因;(4)选择可能分别以ESR1和PGR为靶基因的miRNA100、99a和miRNA378、768-3p,用Real-Time RT-PCR技术验证在体内和体外培养的两个细胞系以及10例人内膜癌标本中其表达的差异性。
结果经鉴定确认Ishikawa来源于Ⅰ型内膜癌,KLE来源于Ⅱ型内膜癌;miRNA芯片筛选出两个细胞系中差异表达的miRNA共126个;基因表达谱芯片筛选出两个细胞系中差异表达的基因共3355个;蛋白质组学分析在两个细胞系中共鉴定出差异表达的蛋白14个;鉴定出的蛋白和免疫组化检测的共18个蛋白中有13个预测到可能以其为靶基因的目标miRNA;Real-Time RT-PCR验证出hsa-miR-100在Ⅰ型组病理标本中的表达显著低于Ⅱ型组,miRNA100、99a、378、768-3p在体外和体内培养的细胞系Ishikawa和KLE中的差异表达基本一致。
结论本实验在两种内膜癌细胞系中首次鉴定出的14个蛋白具有成为内膜癌分型标志的潜力;13个蛋白分别预测出目标miRNA;hsa-miR-100的靶基因很可能为ESR1;本实验所采用的筛选与预测方法能较大的缩窄miRNA靶基因的预测范围。
Objective To identify the differentially expressed miRNA between typeⅠand typeⅡendometrial adenocarcinoma,predict their target gene and validate primarily.
Methods(1) Identified the two endometrial adenocarcinoma cell lines' type by transplanting to nude mice and biopsy,identifying ER,PR,p53 and Ki-67 by immunohistochemistry,and testing their response to estrogen and progesterone.(2) Cultured the two cell lines under the estrogen and progesterone-free circumstance, isolated their total RNA to identify the differentially expressed miRNA and gene by microarray chips;isolated the total protein to perform proteomics analysis.(3) Combined the results from microarray and proteomics,and predicted the differentially expressed miRNAs' target genes by software miRANDA and TargetScan.(4) Validated the expression of miRNA100,99a,378,768-3p who might target ESR1 and PGR in the two cell lines cultured both in vivo and in vitro and ten samples from patients.
Results Confirmed that cell line Ishikawa was from typeⅠendometrial adenocarcinoma and cell line KLE was from typeⅡendometrial adenocarcinoma; identified 126 differentially expressed miRNAs between the two cell lines by miRNA microarray;identified 3355 differentially expressed genes between the two cell lines by gene expression microarray;identified 14 differentially expressed proteins which are novel in endometrial carcinoma between the two cell lines by proteomics;among the 18 proteins from proteomics and immunohistochemstry 13 of them had predicted miRNAs who might target them;Real-Time RT-PCR validated that hsa-miR-100 was significantly down expressed in typeⅠgroup samples than typeⅡgroup,the differential expression of miRNA100,99a,378,768-3p between Ishikawa and KLE was equal in vivo and in vitro.
Conclusions The 14 proteins identified from the two cell lines which are novel in endometrial carcinoma have potential to be markers to identify the endometrial carcinoma's types;13 proteins have been predicted miRNAs who might target them;hsa-miR-100 have great potential to target ESR1;the method we used in this study could be a novel way to limit the the amount of miRNA's predicted target genes to a small number.
方法(1)通过体内成瘤病理学检查、免疫组化检测ER、PR、p53和Ki-67的表达、对雌孕激素作用的反应来鉴定两个细胞系Ishikawa和KLE的分型;(2)无雌孕激素环境下培养细胞系Ishikawa和KLE,提取总RNA进行miRNA芯片检测和基因表达谱芯片检测;提取总蛋白进行蛋白质组学分析;(3)用miRANDA和TargetScan软件结合芯片和蛋白质分析结果,预测差异表达的miRNA可能的靶基因;(4)选择可能分别以ESR1和PGR为靶基因的miRNA100、99a和miRNA378、768-3p,用Real-Time RT-PCR技术验证在体内和体外培养的两个细胞系以及10例人内膜癌标本中其表达的差异性。
结果经鉴定确认Ishikawa来源于Ⅰ型内膜癌,KLE来源于Ⅱ型内膜癌;miRNA芯片筛选出两个细胞系中差异表达的miRNA共126个;基因表达谱芯片筛选出两个细胞系中差异表达的基因共3355个;蛋白质组学分析在两个细胞系中共鉴定出差异表达的蛋白14个;鉴定出的蛋白和免疫组化检测的共18个蛋白中有13个预测到可能以其为靶基因的目标miRNA;Real-Time RT-PCR验证出hsa-miR-100在Ⅰ型组病理标本中的表达显著低于Ⅱ型组,miRNA100、99a、378、768-3p在体外和体内培养的细胞系Ishikawa和KLE中的差异表达基本一致。
结论本实验在两种内膜癌细胞系中首次鉴定出的14个蛋白具有成为内膜癌分型标志的潜力;13个蛋白分别预测出目标miRNA;hsa-miR-100的靶基因很可能为ESR1;本实验所采用的筛选与预测方法能较大的缩窄miRNA靶基因的预测范围。
Objective To identify the differentially expressed miRNA between typeⅠand typeⅡendometrial adenocarcinoma,predict their target gene and validate primarily.
Methods(1) Identified the two endometrial adenocarcinoma cell lines' type by transplanting to nude mice and biopsy,identifying ER,PR,p53 and Ki-67 by immunohistochemistry,and testing their response to estrogen and progesterone.(2) Cultured the two cell lines under the estrogen and progesterone-free circumstance, isolated their total RNA to identify the differentially expressed miRNA and gene by microarray chips;isolated the total protein to perform proteomics analysis.(3) Combined the results from microarray and proteomics,and predicted the differentially expressed miRNAs' target genes by software miRANDA and TargetScan.(4) Validated the expression of miRNA100,99a,378,768-3p who might target ESR1 and PGR in the two cell lines cultured both in vivo and in vitro and ten samples from patients.
Results Confirmed that cell line Ishikawa was from typeⅠendometrial adenocarcinoma and cell line KLE was from typeⅡendometrial adenocarcinoma; identified 126 differentially expressed miRNAs between the two cell lines by miRNA microarray;identified 3355 differentially expressed genes between the two cell lines by gene expression microarray;identified 14 differentially expressed proteins which are novel in endometrial carcinoma between the two cell lines by proteomics;among the 18 proteins from proteomics and immunohistochemstry 13 of them had predicted miRNAs who might target them;Real-Time RT-PCR validated that hsa-miR-100 was significantly down expressed in typeⅠgroup samples than typeⅡgroup,the differential expression of miRNA100,99a,378,768-3p between Ishikawa and KLE was equal in vivo and in vitro.
Conclusions The 14 proteins identified from the two cell lines which are novel in endometrial carcinoma have potential to be markers to identify the endometrial carcinoma's types;13 proteins have been predicted miRNAs who might target them;hsa-miR-100 have great potential to target ESR1;the method we used in this study could be a novel way to limit the the amount of miRNA's predicted target genes to a small number.
引文
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2.Zhao Y,Ransom JF,Li A,Vedantham V,yon Drehle M,Muth AN,Tsuchihsahi T,McManus MT,Schwartz RJ,Srivastava D.Cell 2007;129:1-15.
3.Kumamoto K,Spillare EA,Fujita K,et al.Nutlin-3a activates p53 to both down-regulate inhibitor of growth 2 and up-regulate mir-34a,mir-34b,and mir-34c expression,and induce senescence.Cancer Res.2008 May 1;68(9):3193-203.
4.Tazawa H,Tsuchiya N,Izumiya M,et al.Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells.Proc Natl Acad Sci U S A.2007 Sep 25;104(39):15472-7.
5.Xia L,Zhang D,Du R,et al,.miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells.Int J Cancer.2008 Jul 15;123(2):372-9.
6.Asangani IA,Rasheed SA,Nikolova DA,et al.MieroRNA-21(miR-21) post-transcriptionally downregulates tumor suppressor Pdcd4 and stimulates invasion,intravasation and metastasis in colorectal cancer.Oncogene.2008 Apr 3;27(15):2128-36.Epub 2007 Oct 29.
7.Harris TA,Yamakuchi M,Ferlito M,et al.MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1.Proc Natl Acad Sci U S A.2008 Feb 5;105(5):1516-21.Epub 2008 Jan 28.
8.Lee DY,Deng Z,Wang CH,et al.MieroRNA-378 promotes call survival,tumor growth,and angiogenesis by targeting SuFu and Fus-1 expression.Proc Natl Acad Sci U S A.2007 Dec 18;104(51):20350-5.Epub 2007 Dec 11.
9.Donald E.Kuhn,et al.Experimental Validation of miSNA Targets.Methods.2008January;44(1):47-54.
10.Visone R,Russo L,Pallante P,et al.MicroRNAs(miR)-221 and miR-222,both overexpressed in human thyroid papillary carcinomas,regulate p27Kipl protein levels and cell cycle.Endocr Relat Cancer.2007 Sep;14(3):791-8.
11.Li Y,Qin X,Cui J,Dai Z,et al.Proteome analysis of aflatoxin B1-induced hepatocarcinogenesis in tree shrew(Tupaia bolangeri chinensis) and functional identification of candidate protein peroxiredoxin Ⅱ.Proteomics.2008 Apr;,8(7):1490-501.
12.Memon AA,Chang JW,Oh BR,Yoo YJ,et al.Identification of differentially expressed proteins during human urinary bladder cancer progression.Cancer Detect Prev.2005;29(3):249-55.
13.Soini Y,Kallio JP,Hirvikoski P,et al.Oxidative/nitrosative stress and peroxiredoxin 2 are associated with grade and prognosis of human renal carcinoma.APMIS.2006 May;114(5):329-37.
14.Okochi-Takada E,Nakazawa K,Wakabayashi M,et al.Silencing of the UCHL1 gene in human colorectal and ovarian cancers.Int J Cancer.2006 Sep 15;119(6):1338-44.
15.Seliger B,Fedorushchenko A,Brenner W,et al.Ubiquitin COOH-terminal hydrolase 1:a biomarker of renal cell carcinoma associated with enhanced tumor cell proliferation and migration.Clin Cancer Res.2007 Jan 1;13(1):27-37.
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