MicroRNA-503抑制乳腺癌细胞增殖的作用及其机制研究
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摘要
目的观察miR-503对乳腺癌细胞增殖的影响并探讨其作用机制。方法收集35对乳腺癌及癌旁组织,利用实时荧光定量PCR法检测和比较其miR-503的表达。选择体外培养的乳腺癌T47D细胞系,分别转染miR-503模拟物和miR阴性对照模拟物(miR-NC),构建miR-503和miR-NC过表达细胞系。同时,利用荧光素酶报告基因检测法探索miR-503的下游靶基因。转染miR-503靶基因的真核表达质粒于上述两种T47D细胞系中,通过MTT检测细胞活力变化。结果 miR-503在乳腺癌组织中的表达水平显著低于癌旁组织。过表达miR-503能显著降低乳腺癌T47D细胞的增殖。胰岛素样生长因子1型受体(IGF-1R)是miR-503的靶基因,其在T47D细胞中的蛋白表达水平被miR-503负调控,上调IGF-1R的表达能显著逆转miR-503对T47D细胞增殖的抑制作用。结论 miR-503通过负调控IGF-1R的表达,从而抑制乳腺癌细胞的增殖。
Objective To investigate the effects of miR-503 on the proliferation of breast cancer cells and its mechanism. Methods In the present study, we collected 35 paired breast cancer tissues and matched adjacent tissues, and their miR-503 expressions were detected by real-time fluorescence quantification PCR. T47 D cells were transfected with miR-503 mimic and miR negative controls(miR-NC), respectively. Luciferase assay for candidate miR-503 target gene, insulin-like growth factor 1 receptor(IGF-1R), was performed, and the eukaryotic expression plasmids of this gene were transfected into T47 D cells which were pretreated with miR-503 or miR-NC. Then activities of all these treated cells were measured by MTT. Results Real-time PCR data showed that miR-503 was significantly downregulated in breast cancer tissues as compared to adjacent non-tumor tissues. Overexpression of miR-503 significantly suppressed the proliferation of breast cancer T47 D cells. Bioinformatics analysis and luciferase reporter assay data indicated that insulin-like growth factor 1 receptor(IGF-1R) was a target gene of miR-503, and the protein expression of IGF-1R was negatively mediated by miR-503 in T47 D cells. Moreover, overexpression of IGF-1R remarkably reversed the suppressive effect of miR-503 on T47D cell proliferation, suggesting that the suppressive effect of miR-503 on T47 D cells was directly targeting IGF-1R. Conclusion miR-503 inhibited the proliferation of breast cancer cells through its negative mediation on expression of IGF-1R. The miR-503/IGF-1R signaling may become a potential therapeutic target for breast cancer treatment.
Objective To investigate the effects of miR-503 on the proliferation of breast cancer cells and its mechanism. Methods In the present study, we collected 35 paired breast cancer tissues and matched adjacent tissues, and their miR-503 expressions were detected by real-time fluorescence quantification PCR. T47 D cells were transfected with miR-503 mimic and miR negative controls(miR-NC), respectively. Luciferase assay for candidate miR-503 target gene, insulin-like growth factor 1 receptor(IGF-1R), was performed, and the eukaryotic expression plasmids of this gene were transfected into T47 D cells which were pretreated with miR-503 or miR-NC. Then activities of all these treated cells were measured by MTT. Results Real-time PCR data showed that miR-503 was significantly downregulated in breast cancer tissues as compared to adjacent non-tumor tissues. Overexpression of miR-503 significantly suppressed the proliferation of breast cancer T47 D cells. Bioinformatics analysis and luciferase reporter assay data indicated that insulin-like growth factor 1 receptor(IGF-1R) was a target gene of miR-503, and the protein expression of IGF-1R was negatively mediated by miR-503 in T47 D cells. Moreover, overexpression of IGF-1R remarkably reversed the suppressive effect of miR-503 on T47D cell proliferation, suggesting that the suppressive effect of miR-503 on T47 D cells was directly targeting IGF-1R. Conclusion miR-503 inhibited the proliferation of breast cancer cells through its negative mediation on expression of IGF-1R. The miR-503/IGF-1R signaling may become a potential therapeutic target for breast cancer treatment.
引文
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[20]King H,Aleksic T,Haluska P,et al.Can we unlock the potential of IGF-1R inhibition in cancer therapy?[J].Cancer Treat Rev,2014,40(9):1096-1105.doi:10.1016/j.ctrv.2014.07.004.
[21]Crudden C,Girnita A,Girnita L.Targeting the IGF-1R:The Tale of the Tortoise and the Hare[J].Front Endocrinol(Lausanne),2015,6:64.doi:10.3389/fendo.2015.00064.
[22]Singh P,Alex JM,Bast F.Insulin receptor(IR)and insulinlike growth factor receptor 1(IGF-1R)signaling systems:novel treatment strategies for cancer[J].Med Oncol,2014,31(1):805.doi:10.1007/s12032-013-0805-3.
[23]Dricu A,Kanter L,Wang M,et al.Expression of the insulin-like growth factor 1 receptor(IGF-1R)in breast cancer cells:evidence for a regulatory role of dolichyl phosphate in the transition from an intracellular to an extracellular IGF-1pathway[J].Glycobiology,1999,9(6):571-579.
[24]Dricu A,Kanter L,Wang M,et al.The m RNA expression of IGF-1 and IGF-1R in human breast cancer:association with clinico-pathological parameters[J].Glycobiology,1999,9(6):571-579.
[25]Kucab JE,Dunn SE.Role of IGF-1R in mediating breast cancer invasion and metastasis[J].Breast Dis,2003,17:41-47.
[26]Hou X,Huang F,Macedo LF,et al.Dual IGF-1R/Ins R inhibitor BMS-754807 synergizes with hormonal agents in treatment of estrogen-dependent breast cancer[J].Cancer Res,2011,71(24):7597-7607.doi:10.1158/0008-5472.CAN-11-1080.
[27]Chen Y,Zhu C,Peng Z,et al.Lentivirus-mediated shorthairpin RNA targeting IGF-1R inhibits growth and lymphangiogenesis in breast cancer[J].Oncol Rep,2012,28(5):1778-1784.doi:10.3892/or.2012.1964.
[2]Bièche I,Lidereau R.Genetic alterations in breast cancer[J].Genes Chromosomes Cancer,1995,14(4):227-251.
[3]Ambros V.The functions of animal micro RNAs[J].Nature,.2004,431(7006):350-355.
[4]Serpico D,Molino L,Di Cosimo S.micro RNAs in breast cancer development and treatment[J].Cancer Treat Rev,2014,40(5):595-604.doi:10.1016/j.ctrv.2013.11.002.
[5]Ye JJ,Cao J.Micro RNAs in colorectal cancer as markers and targets:Recent advances[J].World J Gastroenterol,2014,20(15):4288-4299.doi:10.3748/wjg.v20.i15.4288.
[6]Jin L,Wessely O,Marcusson EG,et al.Prooncogenic factors mi R-23b and mi R-27b are regulated by Her2/Neu,EGF,and TNF-αin breast cancer[J].Cancer Res,2013,73(9):2884-2896.doi:10.1158/0008-5472.CAN-12-2162.
[7]Derfoul A,Juan AH,Difilippantonio MJ,et al.Decreased micro RNA-214 levels in breast cancer cells coincides with increased cell proliferation,invasion and accumulation of the Polycomb Ezh2 methyltransferase[J].Carcinogenesis,2011,32(11):1607-1614.doi:10.1093/carcin/bgr184.
[8]Liu H,Song Z,Liao D,et al.mi R-503 inhibits cell proliferation and invasion in glioma by targeting L1CAM[J].Int J Clin Exp Med,2015,8(10):18441-18447.
[9]Chang SW,Yue J,Wang BC,et al.mi R-503 inhibits cell proliferation and induces apoptosis in colorectal cancer cells by targeting E2F3[J].Int J Clin Exp Pathol,2015,8(10):12853-12860.
[10]Long J,Ou C,Xia H,et al.Mi R-503 inhibited cell proliferation of human breast cancer cells by suppressing CCND1expression[J].Tumour Biol,2015,36(11):8697-8702.doi:10.1007/s13277-015-3623-8.
[11]Bartel DP.Micro RNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
[12]Visone R,Pallante P,Vecchione A,et al.Specific micro RNAs are downregulated in human thyroid anaplastic carcinomas[J].Oncogene,2007,26(54):7590-7595.
[13]Zhu J,Zeng Y,Xu C,et al.Expression profile analysis of micro RNAs and downregulated mi R-486-5p and mi R-30a-5p in non-small cell lung cancer[J].Oncol Rep,2015,34(4):1779-1786.doi:10.3892/or.2015.4141.
[14]Wang K,Jia Z,Zou J,et al.Analysis of hsa-mi R-30a-5p expression in human gliomas[J].Pathol Oncol Res,2013,19(3):405-411.doi:10.1007/s12253-012-9593-x.
[15]Kimura S,Naganuma S,Susuki D,et al.Expression of micro RNAs in squamous cell carcinoma of human head and neck and the esophagus:mi R-205 and mi R-21 are specific markers for HNSCC and ESCC[J].Oncol Rep,2010,23(6):1625-1633.
[16]Dai H,Kang B,Zuo D,et al.Effect of mi R-30a-5p on the proliferation,apoptosis,invasion and migration of SMCC-7721 human hepatocellular carcinoma cells[J].Chin J Hepatol,2014,22(12):915-920.doi:10.3760/cma.j.issn.1007-3418.2014.12.008.
[17]Jia Z,Wang K,Wang G,et al.Mi R-30a-5p antisense oligonucleotide suppresses glioma cell growth by targeting SEPT7[J].PLo S One,2013,8(1):e55008.doi:10.1371/journal.pone.0055008.
[18]Wang X,Wang K,Han L,et al.PRDM1 is directly targeted by mi R-30a-5p and modulates the Wnt/β-catenin pathway in a Dkk1-dependent manner during glioma growth[J].Cancer Lett,2013,331(2)211-219.doi:10.1016/j.canlet.2013.01.005.
[19]Baraniskin A,Birkenkamp-Demtroder K,Maghnouj A,et al.Mi R-30a-5p suppresses tumor growth in colon carcinoma by targeting DTL[J].Carcinogenesis,2012,33(4):732-739.doi:10.1093/carcin/bgs020.
[20]King H,Aleksic T,Haluska P,et al.Can we unlock the potential of IGF-1R inhibition in cancer therapy?[J].Cancer Treat Rev,2014,40(9):1096-1105.doi:10.1016/j.ctrv.2014.07.004.
[21]Crudden C,Girnita A,Girnita L.Targeting the IGF-1R:The Tale of the Tortoise and the Hare[J].Front Endocrinol(Lausanne),2015,6:64.doi:10.3389/fendo.2015.00064.
[22]Singh P,Alex JM,Bast F.Insulin receptor(IR)and insulinlike growth factor receptor 1(IGF-1R)signaling systems:novel treatment strategies for cancer[J].Med Oncol,2014,31(1):805.doi:10.1007/s12032-013-0805-3.
[23]Dricu A,Kanter L,Wang M,et al.Expression of the insulin-like growth factor 1 receptor(IGF-1R)in breast cancer cells:evidence for a regulatory role of dolichyl phosphate in the transition from an intracellular to an extracellular IGF-1pathway[J].Glycobiology,1999,9(6):571-579.
[24]Dricu A,Kanter L,Wang M,et al.The m RNA expression of IGF-1 and IGF-1R in human breast cancer:association with clinico-pathological parameters[J].Glycobiology,1999,9(6):571-579.
[25]Kucab JE,Dunn SE.Role of IGF-1R in mediating breast cancer invasion and metastasis[J].Breast Dis,2003,17:41-47.
[26]Hou X,Huang F,Macedo LF,et al.Dual IGF-1R/Ins R inhibitor BMS-754807 synergizes with hormonal agents in treatment of estrogen-dependent breast cancer[J].Cancer Res,2011,71(24):7597-7607.doi:10.1158/0008-5472.CAN-11-1080.
[27]Chen Y,Zhu C,Peng Z,et al.Lentivirus-mediated shorthairpin RNA targeting IGF-1R inhibits growth and lymphangiogenesis in breast cancer[J].Oncol Rep,2012,28(5):1778-1784.doi:10.3892/or.2012.1964.