miR-223-3p靶向上皮细胞转化序列2基因调控胃癌细胞周期和凋亡的相关性研究
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摘要
目的探讨miRNA-223-3p和上皮细胞转化序列2基因(epithelial cell transforming sequence 2 oncogene,ECT2)在胃癌(gastric cancer,GC)细胞周期和凋亡中的作用和机制,并探讨其与GC临床病理因素的相关性及临床意义.方法首先采用实时荧光聚合酶链反应法(real-time polymerase chain reaction,RT-PCR)和蛋白质印迹法检测正常胃黏膜(GSE-1)和GC细胞(SGC-7901,BGC-823)中ECT2和miR-223-3p的表达水平;其次,采用免疫组织化学RT-PCR法检测80例GC患者的癌组织及相应的癌旁组织(距癌组织边缘>5 cm)中ECT2和miR-223-3p的表达情况;miR-223-3p抑制物和模拟物转染SGC-7901细胞,RT-PCR和Western blot检测转染后miR-223-3p和ECT2的表达.最后用流式细胞仪检测转染后细胞周期和凋亡的变化.结果与正常胃黏膜细胞相比,GC细胞中ECT2和miR-223-3p的表达水平均明显升高(P<0.05);免疫组织化学和RT-PCR结果显示,GC组织中ECT2阳性表达率显著高于癌旁组织(P<0.05);两者表达均与GC的分化程度、Lauren分型相关(P<0.05),与TNM分期密切相关(P<0.01),与患者的性别、年龄、肿瘤直径、Bormann分型无明显相关(P>0.05);两者之间表达呈显著正相关(P<0.05);将miR-223-3p类似物转染SGC-7901细胞后ECT2表达上调,转染抑制剂后ECT2表达下调.miR-223-3p抑制物促使肿瘤细胞的G1期阻滞和促进凋亡作用.结论 miR-223-3p是一种与GC细胞周期和凋亡密切相关的miRNA分子,他可以通过影响ECT2的表达来调节GC细胞的细胞周期和凋亡;两者可以作为反映GC生物学行为的有效指标.
AIM To explore the role of miRNA-223-3 p and epithelial cell transforming sequence 2 oncogene(ECT2) in cell cycle and apoptosis of gastric cancer(GC) cells and to analyze their correlation with clinicopathological characteristics.METHODS The expression of ECT2 and miR-223-3 p in normal gastric mucosa cells(GSE-1) and GC cells(SGC-7901 and BGC-823) was detected by real-time fluorescent quantitative PCR and Western blot. Immunohistochemistry and RT-PCR were used to examine the expression of ECT2 and miR-223-3 p in GC tissues and paired adjacent normal tissues, respectively. The correlation between ECT2 and miR-223-3 p expression and clinicopathological characteristics was then analyzed. After miRNA-223-3 p inhibitor and mimic were used to transfect SGC-7901 cells with LipofectamineTM2000,the expression of miRNA-223-3 p and ECT2 was assessed by RT-PCR and Western blot in SGC-7901 cells. After another 24 h culture, the apoptosis rate and cell cycle progression were examined by flow cytometry.RESULTS The expression levels of ECT2 and miR-223-3 p in GC cells were significantly increased as compared with those in normal gastric mucosa cells(P < 0.05 for both).In comparison with tumor adjacent normal tissues, the expression of ECT2 and miR-223-3 p in GC tissues was significantly higher(P < 0.05). The expression of ECT2 and miR-223-3 p was related to histologic differentiation(P < 0.05), Lauren type(P < 0.05), and TNM stage(P <0.01), but not with gender, age, Bormann type, or tumor size(P > 0.05). Transfection with miR-223-3 p mimic up-regulated ECT2 expression, whereas transfection of miR-223-3 p inhibitor downregulated the expression of ECT2. Compared with negative control cells, the apoptosis rate of SGC-7901 cells transfected with miR-223-3 p inhibitor significantly increased(P < 0.05),and the percentage of G1 phase cells also significantly increased in miR-223-3 p inhibitor transfected cells(P< 0.05).CONCLUSION MiR-223-3 p is closely related with cell cycle and apoptosis of gastric cancer cells, and it can regulate the occurrence and development of GC by influencing the expression of ECT2. ECT2 and miR-223-3 p may serve as good factors to indicate the biologic behavior of GC.
AIM To explore the role of miRNA-223-3 p and epithelial cell transforming sequence 2 oncogene(ECT2) in cell cycle and apoptosis of gastric cancer(GC) cells and to analyze their correlation with clinicopathological characteristics.METHODS The expression of ECT2 and miR-223-3 p in normal gastric mucosa cells(GSE-1) and GC cells(SGC-7901 and BGC-823) was detected by real-time fluorescent quantitative PCR and Western blot. Immunohistochemistry and RT-PCR were used to examine the expression of ECT2 and miR-223-3 p in GC tissues and paired adjacent normal tissues, respectively. The correlation between ECT2 and miR-223-3 p expression and clinicopathological characteristics was then analyzed. After miRNA-223-3 p inhibitor and mimic were used to transfect SGC-7901 cells with LipofectamineTM2000,the expression of miRNA-223-3 p and ECT2 was assessed by RT-PCR and Western blot in SGC-7901 cells. After another 24 h culture, the apoptosis rate and cell cycle progression were examined by flow cytometry.RESULTS The expression levels of ECT2 and miR-223-3 p in GC cells were significantly increased as compared with those in normal gastric mucosa cells(P < 0.05 for both).In comparison with tumor adjacent normal tissues, the expression of ECT2 and miR-223-3 p in GC tissues was significantly higher(P < 0.05). The expression of ECT2 and miR-223-3 p was related to histologic differentiation(P < 0.05), Lauren type(P < 0.05), and TNM stage(P <0.01), but not with gender, age, Bormann type, or tumor size(P > 0.05). Transfection with miR-223-3 p mimic up-regulated ECT2 expression, whereas transfection of miR-223-3 p inhibitor downregulated the expression of ECT2. Compared with negative control cells, the apoptosis rate of SGC-7901 cells transfected with miR-223-3 p inhibitor significantly increased(P < 0.05),and the percentage of G1 phase cells also significantly increased in miR-223-3 p inhibitor transfected cells(P< 0.05).CONCLUSION MiR-223-3 p is closely related with cell cycle and apoptosis of gastric cancer cells, and it can regulate the occurrence and development of GC by influencing the expression of ECT2. ECT2 and miR-223-3 p may serve as good factors to indicate the biologic behavior of GC.
引文
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14 Chen Y,Tian P,Liu Y.P53 and Protein Phosphorylation Regulate the Oncogenic Role of Epithelial Cell Transforming2(ECT2).Med Sci Monit 2017;23:3154-3160[PMID:28654632]
15 Justilien V,Ali SA,Jamieson L.ECT2-Dependent rRNASynthesis Is Critical for Lung Cancer Oncogenesis.Cancer Discov 2017;7:Of9[DOI:10.1158/2159-8290.CD-RW2017-025]
16 Justilien V,Fields AP.Ect2 links the PKCiota-Par6alpha complex to Rac1 activation and cellular transformation.Oncogene 2009;28:3597-3607[PMID:19617897 DOI:10.1038/onc.2009.217]
17 Varol N,Konac E,Gurocak OS,Sozen S.The realm of microRNAs in cancers.Mol Biol Rep 2011;38:1079-1089[PMID:20563858 DOI:10.1007/s11033-010-0205-0]
18 Chen CZ,Li L,Lodish HF,Bartel DP.MicroRNAs modulate hematopoietic lineage differentiation.Science 2004;303:83-86[PMID:14657504 DOI:10.1126/science.1091903]
19 Johnnidis JB,Harris MH,Wheeler RT,Stehling-Sun S,Lam MH,Kirak O,Brummelkamp TR,Fleming MD,Camargo FD.Regulation of progenitor cell proliferation and granulocyte function by microRNA-223.Nature 2008;451:1125-1129[PMID:18278031 DOI:10.1038/nature06607]
20 Zhou X,Jin W,Jia H,Yan J,Zhang G.MiR-223 promotes the cisplatin resistance of human gastric cancer cells via regulating cell cycle by targeting FBXW7.J Exp Clin Cancer Res 2015;34:28[PMID:25888377 DOI:10.1186/s13046-015-0145-6]
21 Fang G,Liu J,Wang Q,Huang X,Yang R,Pang Y,Yang M.MicroRNA-223-3p Regulates Ovarian Cancer Cell Proliferation and Invasion by Targeting SOX11 Expression.Int J Mol Sci 2017;18:pii E1208[PMID:28587313 DOI:10.3390/ijms18061208]
22 Fassan M,Saraggi D,Balsamo L,Realdon S,Scarpa M,Castoro C,Coati I,Salmaso R,Farinati F,Guzzardo V,Arcidiacono D,Munari G,Gasparini P,Veronese N,Luchini C,Valeri N,Rugge M.Early miR-223 Upregulation in Gastroesophageal Carcinogenesis.Am J Clin Pathol 2017;147:301-308[PMID:28395057 DOI:10.1093/ajcp/aqx004]
23 Li R,Wu S,Chen X,Xu H,Teng P,Li W.miR-223/FBW7axis regulates doxorubicin sensitivity through epithelial mesenchymal transition in non-small cell lung cancer.Am JTransl Res 2016;8:2512-2524[PMID:27398136]
24 Xu J,Yao Q,Hou Y,Xu M,Liu S,Yang L,Zhang L,Xu H.MiR-223/Ect2/p21 signaling regulates osteosarcoma cell cycle progression and proliferation.Biomed Pharmacother 2013;67:381-386[PMID:23601845 DOI:10.1016/j.biopha.2013.03.013]
2 Shrestha S,Hsu SD,Huang WY,Huang HY,Chen W,Weng SL,Huang HD.A systematic review of microRNAexpression profiling studies in human gastric cancer.Cancer Med 2014;3:878-888[PMID:24902858 DOI:10.1002/cam4.246]
3 Ishimoto T,Baba H,Izumi D,Sugihara H,Kurashige J,Iwatsuki M,Tan P.Current perspectives toward the identification of key players in gastric cancer microRNAdysregulation.Int J Cancer 2016;138:1337-1349[PMID:26041092 DOI:10.1002/ijc.29627]
4 Boldrup L,Gu X,Coates PJ,Norberg-Spaak L,Fahraeus R,Laurell G,Wilms T,Nylander K.Gene expression changes in tumor free tongue tissue adjacent to tongue squamous cell carcinoma.Oncotarget 2017;8:19389-19402[PMID:28038473 DOI:10.18632/oncotarget.14288]
5 Vazquez-Mena O,Medina-Martinez I,Juárez-Torres E,Barrón V,Espinosa A,Villegas-Sepulveda N,GómezLaguna L,Nieto-Martínez K,Orozco L,Roman-Basaure E,Mu駉z Cortez S,Borges Iba馿z M,Venegas-Vega C,Guardado-Estrada M,Rangel-López A,Kofman S,Berumen J.Amplified genes may be overexpressed,unchanged,or downregulated in cervical cancer cell lines.PLoS One 2012;7:e32667[PMID:22412903 DOI:10.1371/journal.pone.0032667]
6 Chen J,Xia H,Zhang X,Karthik S,Pratap SV,Ooi LL,Hong W,Hui KM.ECT2 regulates the Rho/ERK signalling axis to promote early recurrence in human hepatocellular carcinoma.J Hepatol 2015;62:1287-1295[PMID:25617497DOI:10.1016/j.jhep.2015.01.014]
7 Huff LP,Decristo MJ,Trembath D,Kuan PF,Yim M,Liu J,Cook DR,Miller CR,Der CJ,Cox AD.The Role of Ect2 Nuclear RhoGEF Activity in Ovarian Cancer Cell Transformation.Genes Cancer 2013;4:460-475[PMID:24386507 DOI:10.1177/1947601913514851]
8 Backes C,Kehl T,St鯿kel D,Fehlmann T,Schneider L,Meese E,Lenhof HP,Keller A.miRPathDB:a new dictionary on microRNAs and target pathways.Nucleic Acids Res 2017;45:D90-D96[PMID:27742822 DOI:10.1093/nar/gkw926]
9 Remmele W,Stegner HE.Recommendation for uniform definition of an immunoreactive score(IRS)for immunohistochemical estrogen receptor detection(ER-ICA)in breast cancer tissue.Pathologe 1987;8:138-140[PMID:3303008]
10 Miki T,Smith CL,Long JE,Eva A,Fleming TP.Oncogene ect2is related to regulators of small GTP-binding proteins.Nature1993;362:462-465[PMID:8464478 DOI:10.1038/362462a0]
11 Fields AP,Justilien V.The guanine nucleotide exchange factor(GEF)Ect2 is an oncogene in human cancer.Adv Enzyme Regul 2010;50:190-200[PMID:19896966 DOI:10.1016/j.advenzreg.2009.10.010]
12 Jing J,Chen L,Fu HY,Fan K,Yao Q,Ge YF,Lu JC,Yao B.Annexin V-induced rat Leydig cell proliferation involves Ect2 via RhoA/ROCK signaling pathway.Sci Rep 2015;5:9437[PMID:25807302 DOI:10.1038/srep09437]
13 Justilien V,Lewis KC,Murray NR,Fields AP.Oncogenic Ect2 signaling regulates rRNA synthesis in NSCLC.Small GTPases 2017 Jun 28.[Epub ahead of print][PMID:28657426DOI:10.1080/21541248.2017.1335274]
14 Chen Y,Tian P,Liu Y.P53 and Protein Phosphorylation Regulate the Oncogenic Role of Epithelial Cell Transforming2(ECT2).Med Sci Monit 2017;23:3154-3160[PMID:28654632]
15 Justilien V,Ali SA,Jamieson L.ECT2-Dependent rRNASynthesis Is Critical for Lung Cancer Oncogenesis.Cancer Discov 2017;7:Of9[DOI:10.1158/2159-8290.CD-RW2017-025]
16 Justilien V,Fields AP.Ect2 links the PKCiota-Par6alpha complex to Rac1 activation and cellular transformation.Oncogene 2009;28:3597-3607[PMID:19617897 DOI:10.1038/onc.2009.217]
17 Varol N,Konac E,Gurocak OS,Sozen S.The realm of microRNAs in cancers.Mol Biol Rep 2011;38:1079-1089[PMID:20563858 DOI:10.1007/s11033-010-0205-0]
18 Chen CZ,Li L,Lodish HF,Bartel DP.MicroRNAs modulate hematopoietic lineage differentiation.Science 2004;303:83-86[PMID:14657504 DOI:10.1126/science.1091903]
19 Johnnidis JB,Harris MH,Wheeler RT,Stehling-Sun S,Lam MH,Kirak O,Brummelkamp TR,Fleming MD,Camargo FD.Regulation of progenitor cell proliferation and granulocyte function by microRNA-223.Nature 2008;451:1125-1129[PMID:18278031 DOI:10.1038/nature06607]
20 Zhou X,Jin W,Jia H,Yan J,Zhang G.MiR-223 promotes the cisplatin resistance of human gastric cancer cells via regulating cell cycle by targeting FBXW7.J Exp Clin Cancer Res 2015;34:28[PMID:25888377 DOI:10.1186/s13046-015-0145-6]
21 Fang G,Liu J,Wang Q,Huang X,Yang R,Pang Y,Yang M.MicroRNA-223-3p Regulates Ovarian Cancer Cell Proliferation and Invasion by Targeting SOX11 Expression.Int J Mol Sci 2017;18:pii E1208[PMID:28587313 DOI:10.3390/ijms18061208]
22 Fassan M,Saraggi D,Balsamo L,Realdon S,Scarpa M,Castoro C,Coati I,Salmaso R,Farinati F,Guzzardo V,Arcidiacono D,Munari G,Gasparini P,Veronese N,Luchini C,Valeri N,Rugge M.Early miR-223 Upregulation in Gastroesophageal Carcinogenesis.Am J Clin Pathol 2017;147:301-308[PMID:28395057 DOI:10.1093/ajcp/aqx004]
23 Li R,Wu S,Chen X,Xu H,Teng P,Li W.miR-223/FBW7axis regulates doxorubicin sensitivity through epithelial mesenchymal transition in non-small cell lung cancer.Am JTransl Res 2016;8:2512-2524[PMID:27398136]
24 Xu J,Yao Q,Hou Y,Xu M,Liu S,Yang L,Zhang L,Xu H.MiR-223/Ect2/p21 signaling regulates osteosarcoma cell cycle progression and proliferation.Biomed Pharmacother 2013;67:381-386[PMID:23601845 DOI:10.1016/j.biopha.2013.03.013]