Please wait a minute...
Journal of ZheJiang University(Medical Science)  2017, Vol. 46 Issue (4): 349-356    DOI: 10.3785/j.issn.1008-9292.2017.08.02
    
Effect of microRNA-29b on proliferation and migration of breast cancer cells and its molecular mechanism
JIANG Yiqian1,2, GUO Qingmin2, GU Jianzhong3, XU Xiaoping2, AN Suhong2, SU Fang2, BAO Yanhong2, HUANG Changxin2, GUAN Xiaoxiang1
1. Jinling Clinical Medical College, Nanjing Medical University, Nanjing 210002, China;
2. Center of Oncology, Xiaoshan First people's Hospital Affiliated to the Medical College, Hangzhou Normal University, Hangzhou 311200, China;
3. Department of Oncology, the First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou 310006, China
Download:   PDF(4089KB)
Export: BibTeX | EndNote (RIS)      

Abstract  

Objective:To investigate the effects of microRNA(miRNA)-29b on the proliferation and migration of breast cancer cells and its molecular mechanism. Methods:The recombinant lentiviral expression vector (lenti-miRNA-29b) was constructed and transfected into 293T cells to obtain lentivirus particles that were used to infect breast cancer MCF-7 cells. Transfection efficiency of lenti-miRNA-29b in MCF-7 cells was identified by the expression of green fluorescent protein (GFP). The expression of miRNA-29b was detected by real-time PCR. The cell proliferation and migration were detected by CCK8 assay and Transwell assay, respectively. The bioinformatics softwares were used to predict and screen the downstream target genes regulated by miRNA-29b, which were verified by double luciferase reporter gene assay, RT-PCR and Western blot. The effects of screened target gene RTKN on the growth and migration of MCF-7 cells were verified by RTKN siRNA. Results:Recombinant lentiviral expression vector of miRNA-29b were successfully constructed. About 90% and 60% of the breast cancer cells showed green fluorescence in lenti-miRNA-29b and lenti-miRNA-NC groups, respectively. The expression of miRNA-29b in lenti-miRNA-29b group increased significantly compared with the lenti-miRNA-NC group and blank control group (all P<0.05); the proliferation and migration ability of MCF-7 cells significantly reduced compared with the control group (all P<0.05). The screening with bioinformatics softwares found that the 3'UTR coding region RTKN had the binding site to miRNA-29b; the dual luciferase reporter gene assay showed that the luciferase activity decreased significantly after the MCF-7 cells were co-transfected with wild type RTKN-WT-3'UTR and miRNA-29b mimics report gene vector (P<0.05). The RTKN proteins in MCF-7 cells were significantly decreased after transfection with siRNA-RTKN, and the proliferation and migration ability of MCF-7 cells were significantly reduced (all P<0.05). Conclusion:MiRNA-29b can inhibit the proliferation, invasion and metastasis of breast cancer cells by inhibiting the expression of RTKN.



Key wordsMicroRNAs      Breast neoplasms/physiopathology      Lentivirus      Transfection      Polymerase chain reaction      Cell proliferation      Neoplasm metastasis      Tumor cells, cultured     
Received: 05 May 2017      Published: 25 August 2017
CLC:  R737.9  
Cite this article:

JIANG Yiqian, GUO Qingmin, GU Jianzhong, XU Xiaoping, AN Suhong, SU Fang, BAO Yanhong, HUANG Changxin, GUAN Xiaoxiang. Effect of microRNA-29b on proliferation and migration of breast cancer cells and its molecular mechanism. Journal of ZheJiang University(Medical Science), 2017, 46(4): 349-356.

URL:

http://www.zjujournals.com/xueshu/med/10.3785/j.issn.1008-9292.2017.08.02     OR     http://www.zjujournals.com/xueshu/med/Y2017/V46/I4/349


微小RNA-29b对乳腺癌细胞增殖和迁移的影响及其分子生物学机制

目的:研究微小RNA(miRNA)-29b对乳腺癌细胞增殖和迁移的影响及其分子生物学机制。方法:构建miRNA-29b重组慢病毒表达载体lenti-miRNA-29b转染293T细胞获得重组慢病毒,提纯慢病毒颗粒感染乳腺癌细胞MCF-7。通过绿色荧光蛋白肿瘤细胞比例来验证转染情况;实时定量PCR检测miRNA-29b的表达水平;CCK-8实验和Transwell实验检测miRNA-29b过表达后乳腺癌细胞生物学行为的变化。采用生物信息学软件对miRNA-29b调控的下游靶基因进行预测和筛选,使用双荧光素酶检测、实时定量PCR和蛋白质印迹法进行验证。对筛选出的miRNA-29b调控的下游靶基因RTKN通过siRNA-RTKN验证其对MCF-7细胞的增殖和迁移的影响。结果:成功构建了miRNA-29b的重组慢病毒表达载体。感染乳腺癌细胞MCF-7后,lenti-miRNA-29b组和空载体转染组分别有90%和60%左右的细胞出现绿色荧光;lenti-miRNA-29b组miRNA-29b表达量较空载体转染组和空白对照组显著增加(均P<0.05),MCF-7细胞的增殖和迁移能力较空载体转染组显著减弱(均P<0.05)。RTKN的基因编码3'UTR区中具有miRNA-29b的结合位点;野生型RTKN-WT-3'UTR报告基因载体与lenti-miRNA-29b共转染MCF-7细胞后可使荧光素酶的活性显著下降(P<0.05)。转染siRNA-RTKN后,MCF-7细胞中RTKN蛋白的水平显著下降,细胞增殖和迁移能力显著减弱(均P<0.05)。结论:miRNA-29b能够通过抑制RTKN的表达来抑制乳腺癌细胞的增殖和迁移能力。


关键词: 微RNAs,  乳腺肿瘤/病理生理,  慢病毒属,  转染,  聚合酶链反应,  细胞增殖,  肿瘤转移,  肿瘤细胞,  培养的 

[1] CORBIN R, OLSSON-CARTER K, SLACK F. The role of microRNAs in synaptic development and function[J]. BMB Rep,2009,42(3):131-135.
[2] LAGOS-QUINTANA M, RAUHUT R, LENDECKEL W, et al. Identification of novel genes coding for small expressed RNAs[J]. Science,2001,294(5543):853-858.
[3] PENG J, ZARNESCU D C, CEMAN S, et al. Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway[J]. Nat Neurosci,2004,7(2):113-117.
[4] ZHANG Y, ZHAO F J, CHEN L L, et al. MiR-373 targeting of the Rab22a oncogene suppresses tumor invasion and metastasis in ovarian cancer[J]. Oncotarget,2014,5(23):12291-12303.
[5] TEOH S L, DAS S. The role of microRNAs in diagnosis, prognosis, metastasis and resistant cases in breast cancer[J]. Curr Pharm Des,2017,23(12):1845-1859.
[6] BERTOLI G, CAVA C, CASTIGLIONI I. MicroRNAs:new biomarkers for diagnosis, prognosis, therapy prediction and therapeutic tools for breast cancer[J]. Theranostics,2015,5(10):1122-1143.
[7] HUANG W. MicroRNAs:biomarkers, diagnostics, and therapeutics[J]. Methods Mol Biol,2017,1617:57-67.
[8] CHUA J H, ARMUGAM A, JEYASEELAN K. MicroRNAs:biogenesis, function and applications[J]. Curr Opin Mol Ther,2009,11(2):189-199.
[9] DOSTIE J, MOURELATOS Z, YANG M, et al. Numerous microRNPs in neuronal cells containing novel microRNAs[J]. RNA,2003,9(2):180-186.
[10] LANG N, LIU M, TANG Q L, et al. Effects of microRNA-29 family members on proliferation and invasion of gastric cancer cell lines[J]. Chin J Cancer,2010,29(6):603-610.
[11] EYHOLZER M, SCHMID S, WILKENS L, et al. The tumour-suppressive miR-29a/b1 cluster is regulated by CEBPA and blocked in human AML[J]. Br J Cancer,2010,103(2):275-284.
[12] 陈万青,郑荣寿.中国女性乳腺癌发病、死亡和生存状况[J].中国肿瘤临床,2015,42(13):668-674. CHEN Wanqing, ZHENG Rongshou. Incidence,mortality and survival analysis of breast cancer in China[J]. Chinese Journal of Clinical Oncology,2015,42(13):668-674. (in Chinese)
[13] 郑莹,吴春晓,张敏璐.乳腺癌在中国的流行状况和疾病特征[J].中国癌症杂志,2013,23(8):561-569. ZHENG Ying, WU Chunxiao, ZHANG Minlu. The epidemic and characteristics of female breast cancer in China[J]. China Oncology,2013,23(8):561-569. (in Chinese)
[14] BARTEL D P. MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell,2004,116(2):281-297.
[15] MISKIEWICZ J, TOMCZYK K, MICKIEWICZ A, et al. Bioinformatics study of structural patterns in plant microRNA precursors[J]. Biomed Res Int,2017,2017:6783010.
[16] ZHANG B, PAN X, COBB G P, et al. Plant microRNA:a small regulatory molecule with big impact[J]. Dev Biol,2006,289(1):3-16.
[17] GARZON R, HEAPHY C E, HAVELANGE V, et al. MicroRNA 29b functions in acute myeloid leukemia[J]. Blood,2009,114(26):5331-5341.
[18] MOTT J L, KURITA S, CAZANAVE S C, et al. Transcriptional suppression of mir-29b-1/mir-29a promoter by c-Myc, hedgehog, and NF-kappaB[J]. J Cell Biochem,2010,110(5):1155-1164.
[19] SHAH N R, CHEN H. MicroRNAs in pathogenesis of breast cancer:Implications in diagnosis and treatment[J]. World J Clin Oncol,2014,5(2):48-60.
[20] YAN B, GUO Q, FU F J, et al. The role of miR-29b in cancer:regulation, function, and signaling[J]. Onco Targets Ther,2015,8:539-548.
[21] WANG C, BIAN Z, WEI D, et al. miR-29b regulates migration of human breast cancer cells[J]. Mol Cell Biochem,2011,352(1-2):197-207.
[22] 范旭龙,吴爱国.微小RNA 29在乳腺癌中的研究进展[J/CD].中华乳腺病杂志:电子版,2013,7(5):355-359. FAN Xulong, WU Aiguo. Research progress on microRNA 29 in breast cancer[J/CD]. Chinese Journal of Breast Disease (Electronic Version),2013,7(5):355-359. (in Chinese)
[23] DENG X, CAO M, ZHANG J, et al. Hyaluronic acid-chitosan nanoparticles for co-delivery of MiR-34a and doxorubicin in therapy against triple negative breast cancer[J]. Biomaterials,2014,35(14):4333-4344.
[24] WANG Y, ZHANG X, LI H, et al. The role of miRNA-29 family in cancer[J]. Eur J Cell Biol,2013,92(3):123-128.
[25] ZHANG K, ZHANG C, LIU L, et al. A key role of microRNA-29b in suppression of osteosarcoma cell proliferation and migration via modulation of VEGF[J]. Int J Clin Exp Pathol,2014,7(9):5701-5708.
[26] ZHANG W, QIAN J X, YI H L, et al. The microRNA-29 plays a central role in osteosarcoma pathogenesis and progression[J]. Mol Biol (Mosk),2012,46(4):622-627.
[27] WANG C, GAO C, ZHUANG J L, et al. A combined approach identifies three mRNAs that are down-regulated by microRNA-29b and promote invasion ability in the breast cancer cell line MCF-7[J]. J Cancer Res Clin Oncol,2012,138(12):2127-2136.
[28] CHOU J, LIN J H, BRENOT A, et al. GATA3 suppresses metastasis and modulates the tumour microenvironment by regulating microRNA-29b expression[J]. Nat Cell Biol,2013,15(2):201-213.

[1] BIAN Wei, YAO Yuan, ZHANG Haijin, YE Xiaoqian, ZHANG Minming. Application of CT scan in diagnosis of pathological type and origin of metastatic ovarian tumors[J]. Journal of ZheJiang University(Medical Science), 2017, 46(5): 546-551.
[2] LI Yu, WANG Yueyue, WANG Haifeng, ZHANG Lingyu, DING Yongxing, CHEN Sulian, YANG Qingling, CHEN Changjie. Effects of lncRNA RP11-770J1.3 and TMEM25 expression on paclitaxel resistance in human breast cancer cells[J]. Journal of ZheJiang University(Medical Science), 2017, 46(4): 364-370.
[3] WANG Haifeng, CHEN Tiantian, WANG Yueyue, LI Yu, ZHANG Lingyu, DING Yongxing, CHEN Sulian, WANG Wenrui, YANG Qingling, CHEN Changjie. CXC chemokine receptor 4 regulates breast cancer cell cycle through S phase kinase associated protein 2[J]. Journal of ZheJiang University(Medical Science), 2017, 46(4): 357-363.
[4] TIAN Hua, CHEN Yang, ZHAO Jiangang, LIU Daren, LIANG Gang, GONG Weihua, CHEN Li, WU Yulian. Effects of siRNAs targeting CD97 immune epitopes on biological behavior in breast cancer cell line MDA-MB231[J]. Journal of ZheJiang University(Medical Science), 2017, 46(4): 341-348.
[5] CHEN Dahua, LI Youming. Construction of all-in-one CRISPR/Cas9 vector system targeting miR-101a gene in mouse hepatic cell line AML12[J]. Journal of ZheJiang University(Medical Science), 2017, 46(4): 427-432.
[6] ZHANG Wei, LAI Lihua, WANG Qingqing. Effects of myeloid specific deficiency of FBXW7 on lung metastasis of murine melanoma[J]. Journal of ZheJiang University(Medical Science), 2017, 46(2): 111-117.
[7] WANG Hebin, LI Yang, LIU Xingang, ZHOU Jun, WANG Qingqing, TANG Guping. Preparation, characterization and cytology study of Pluronic-PEI micelles[J]. Journal of ZheJiang University(Medical Science), 2017, 46(2): 127-133.
[8] ZHANG Wei,LAI Lihua,WANG Qingqing. Effects of myeloid specific deficiency of FBXW7 on lung metastasis of murine melanoma[J]. Journal of ZheJiang University(Medical Science), 2017, 46(2): 111-117.
[9] WANG Hebin,LI Yang,LIU Xingang,ZHOU Jun,WANG Qingqing,TANG Guping. Preparation, characterization and cytology study of Pluronic-PEI micelles[J]. Journal of ZheJiang University(Medical Science), 2017, 46(2): 127-133.
[10] YANG Xiaohong, YANG Kun, LIAO Li, JIN Yan. Effect of miR-705 on osteogenic differentiation of mouse embryo osteoblast precursor cells MC3T3-E1[J]. Journal of ZheJiang University(Medical Science), 2016, 45(6): 575-580.
[11] CAO Peng, LENG Dongjin, LI Ying, ZHANG Ziwei, LIU Lei, LI Xiaoyan. Progress on anti-tumor molecular mechanisms of dihydroartemisinin[J]. Journal of ZheJiang University(Medical Science), 2016, 45(5): 501-507.
[12] WU Zhihua, JING Min, LIANG Hanying, YANG Rong, HUANG Yaping, CHEN Xiaoming, HU Jianhua, FAN Jun. T cell receptor β-chain CDR3 spectratyping and cytomegalovirus activation in allogeneic hematopoietic stem cell transplant recipients[J]. Journal of ZheJiang University(Medical Science), 2016, 45(5): 515-521.
[13] CHEN Xiaojing, XU Junfen, YE Jing, CHENG Xiaodong, XIE Xing, LYU Weiguo. Expression of miR-let-7e-3p in cervical intraepithelial neoplasm and cervix carcinoma and its clinical significance[J]. Journal of ZheJiang University(Medical Science), 2016, 45(4): 342-348.
[14] YANG Suwen, WANG Wei, JIN Hong, ZHONG Yuhong, XIE Xinyou. Expression of microRNA-221/222 in patients with monoclonal gammopathy of undetermined significance and multiple myeloma[J]. Journal of ZheJiang University(Medical Science), 2016, 45(4): 371-378.
[15] LOU Pengrong, SUN Xiaonan, ZHOU Jundong, ZOU Shitao. Effect of RAD18-siRNA on proliferation and chemotherapy sensitivity of human esophageal squamous cell carcinoma ECA-109 cells[J]. Journal of ZheJiang University(Medical Science), 2016, 45(4): 364-370.