miR-17-5p在大鼠垂体泌乳素腺瘤MMQ细胞耐药中的作用
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摘要
目的:探讨microRNA-17-5p(mi R-17-5p)在大鼠垂体泌乳素腺瘤MMQ细胞耐药中的作用及相关机制。方法:利用miR-17-5p类似物及拮抗物分别过表达和抑制大鼠垂体泌乳素腺瘤MMQ细胞中miR-17-5p的表达,并采用实时荧光定量PCR(RT-q PCR)法进行检测。利用CCK-8法检测miR-17-5p表达调控前后细胞增殖以及对卡麦角林(CAB)治疗反应的变化。通过生物信息学方法预测miR-17-5p的靶点基因,然后采用荧光素酶报告检测法进行验证,继而用qRT-PCR及Western blot法检测miR-17-5p对靶基因表达的影响。慢病毒细胞转染过表达MMQ细胞中的PTEN基因,用Western blot检测PTEN的表达水平,并采用CCK-8法检测PTEN过表达对MMQ细胞耐药性的影响。结果:mi R-17-5p过表达可促进MMQ细胞的增殖,而miR-17-5p敲减则能抑制MMQ细胞的增殖。mi R-17-5p敲减能上调MMQ细胞对CAB治疗的敏感性,相反,miR-17-5p过表达则显著降低MMQ细胞对CAB治疗的敏感性。生物信息学方法预测PTEN为miR-17-5p潜在作用靶点,荧光素酶报告检测法进一步验证了两者的相互作用关系,同时miR-17-5p过表达的MMQ细胞中PTEN mRNA及蛋白表达量显著下降,而miR-17-5p敲减的MMQ细胞中PTEN mRNA及蛋白表达量显著上升。PTEN表达上调能部分逆转miR-17-5p过表达引起的MMQ细胞对CAB治疗敏感性降低。结论:miR-17-5p可通过下调PTEN的表达促进MMQ细胞增殖并降低其对CAB治疗的反应。
Objective: To study the roles of microRNA-17-5p may play in the resistance of MMQ cells to dopamine agonists. Methods: MMQ cells were either treated with miR-17-5p mimics or miR-17-5p inhibitor to regulate the expression of miR-17-5p in vitro qPCR was used to verify the transfection efficiency. After treatment, cell viability and its response to CAB was determined with CCK-8 assays. The target gene of miR-17-5p was predicted and confirmed via bioinformatics analysis and luciferase reporter assays. The expression of PTEN was analyzed by qPCR and Western Blotting. In addition we deployed CCK-8 assay to evaluate the response to CAB of MMQ cells which were initially treated with lentiviral vector containing PTEN. Results: Overexpression of miR-17-5p could promote the proliferation of MMQ cells and suppress CAB cytotoxicity. On the contrary, down-regulated of the expression of miR-17-5p could effectively inhibit proliferation and boost susceptibility to CAB in MMQ cells. The bioinformatics database identified the potential target of miR-17-5p and the luciferase reporter assay confirmed that PTEN was the direct target of miR-17-5p. While the expression of PTEN was down-regulated in miR-17-5p over-expressed MMQ cells, the expression of PTEN was up-regulated in miR-17-5p knockdown MMQ cells. Furthermore, PTEN over-expression was able to reverse the drug resistance induced by miR-17-5p on CAB treatment. Conclusion: miR-17-5p promotes MMQ proliferation and mediates CAB resistance by targeting PTEN.
Objective: To study the roles of microRNA-17-5p may play in the resistance of MMQ cells to dopamine agonists. Methods: MMQ cells were either treated with miR-17-5p mimics or miR-17-5p inhibitor to regulate the expression of miR-17-5p in vitro qPCR was used to verify the transfection efficiency. After treatment, cell viability and its response to CAB was determined with CCK-8 assays. The target gene of miR-17-5p was predicted and confirmed via bioinformatics analysis and luciferase reporter assays. The expression of PTEN was analyzed by qPCR and Western Blotting. In addition we deployed CCK-8 assay to evaluate the response to CAB of MMQ cells which were initially treated with lentiviral vector containing PTEN. Results: Overexpression of miR-17-5p could promote the proliferation of MMQ cells and suppress CAB cytotoxicity. On the contrary, down-regulated of the expression of miR-17-5p could effectively inhibit proliferation and boost susceptibility to CAB in MMQ cells. The bioinformatics database identified the potential target of miR-17-5p and the luciferase reporter assay confirmed that PTEN was the direct target of miR-17-5p. While the expression of PTEN was down-regulated in miR-17-5p over-expressed MMQ cells, the expression of PTEN was up-regulated in miR-17-5p knockdown MMQ cells. Furthermore, PTEN over-expression was able to reverse the drug resistance induced by miR-17-5p on CAB treatment. Conclusion: miR-17-5p promotes MMQ proliferation and mediates CAB resistance by targeting PTEN.
引文
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[20]RAFTOPOULOU M,ETIENNE-MANNEVILLE S,SELFA,et al.Regulation of cell migration by the C2 domain of the tumor suppressor PScience TEN[J].Science,2004,303(5661):1179-1181.
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[22]HU Y,XU S,JIN W,et al.Effect of the PTEN gene on adhesion,invasion and metastasis of osteosarcoma cells[J].Oncol Rep,2014,32(4):1741-1747.
[23]CHEN MW,WU XJ.SLC25A22 promotes proliferation and metastasis of osteosarcoma cells via the PTEN signaling pathway[J].Technol Cancer Res Trest,2018,17:1533033818811143.
[24]TIAN K,DI R,WANG L.MicroRNA-23a enhances migration and invasion through PTEN in osteosarcoma[J].Cancer Gene Ther,2015,22(7):351-359.
[25]金晓昇,安慧敏,黄智铭.生存素和PTEN在良恶性胃溃疡中的表达[J].温州医科大学学报,2017,47(6):456-458.
[2]WU Z B,YU C J,SU Z P,et al.Bromocriptine treatment of invasive giant prolactinomas involving the cavernoussinus:results of a long-term follow up[J].J Neurosurg,2006,104(1):54-61.
[3]COLAO A,DI SARNO A,GUERRA E,et al.Predictors of remission of hyperprolactinaemia after long-term withdrawal of cabergoline therapy[J].Clin Endocrinol(Oxf),2007,67(3):426-433.
[4]MOLITCH M E.Pituitary gland:can prolactinomas be cured medically?[J].Nat Rev Endocrinol,2010,6(4):186-188.
[5]WU Z R,ZHANG Y,CAI L,et al.Long-term clinical outcomes of invasive giant prolactinomas after a mean ten-year followup[J].Int J Endocrinol,2016,2016:8580750.
[6]MOLITCH M E.Dopamine resistance of prolactinomas[J].Pituitary,2003,6(1):19-27.
[7]ADAMS B D,PARSONS C,WALKER L,et al.Targeting noncoding RNAs in disease[J].J Clin Invest,2017,127(3):761-771.
[8]ZENG H C,BAE Y,DAWSON B C,et al.MicroRNA miR-23a cluster promotes osteocyte differentiation by regulating TGF-beta signalling in osteoblasts[J].Nat Commun,2017,8:15000.
[9]ZHANG W,XU J,SHI Y,et al.The novel role of miRNAs for tamoxifen resistance in human breast cancer[J].Cell Mol Life Sci,2015,72(13):2575-2584.
[10]DUAN Z,GAO Y,SHEN J,et al.MiR-15b modulates multidrug resistance in human osteosarcoma in vitro and in vivo[J].Mol Oncol,2017,11(2):151-166.
[11]NEESSE A,GRESS T M.Emerging role of microRNAs to tackle drug resistance in pancreatic cancer[J].Gut,2015,64(12):1842-1843.
[12]AHMAD N,HAIDER S,JAGANNATHAN S,et al.MicroRNA theragnostics for the clinical management of multiple myeloma[J].Leukemia,2014,28(4):732-738.
[13]FANINI F,FABBRI M.MicroRNAs and cancer resistance:a new molecular plot[J].Clin Pharmacol Ther,2016,99(5):485-493.
[14]XI X P,ZHUANG J,TENG M J,et al.MicroRNA-17 induces epithelial-mesenchymal transition consistent with the cancer stem cell phenotype by regulating CYP7B1 expression in colon cancer[J].Int J Mol Med,2016,38(2):499-506.
[15]WU Z B,LI W Q,LIN S J,et al.MicroRNA expression profile of bromocriptine-resistant prolactinomas[J].Mol Cell Endocrinol,2014,395(1-2):10-18.
[16]赵晓蕾,蔡林,邓洲铭.microRNA与骨肉瘤的研究进展[J].中国骨与关节杂志,2014,3(10):773-778.
[17]JIA J G,ZHAN D K,LI J,et al.The contrary functions of lncRNA HOTAIR/miR-17-5p/PTEN axis and Shenqifuzheng injection on chemosensitivity of gastric cancer cells[J].J Cell Mol Med,2018,23(1):656-669.
[18]SAEED NOOROLYAI,AHAD MOKHTARZADEH,EL-HAM BAGHBANI,et al.The role of microRNAs involved in PI3-kinase signaling pathway in colorectal cancer[J].JCell Physiol,2018,2018:1-10.
[19]TSUGAWA K,JONES M K,SUGIMACHI K,et al.Biological role of phosphatase PTEN in cancer and tissue injury healing[J].Front Biosci,2002,7:e245-251.
[20]RAFTOPOULOU M,ETIENNE-MANNEVILLE S,SELFA,et al.Regulation of cell migration by the C2 domain of the tumor suppressor PScience TEN[J].Science,2004,303(5661):1179-1181.
[21]GILDEA J J,HERLEVSEN M,HARDING M A,et al.PTEN can inhibit in vitro organotypic and in vivo orthotopic invasion of human bladder cancer cells even in the absence of its lipid phosphatase activity[J].Oncogene,2004,23(40):6788-6797.
[22]HU Y,XU S,JIN W,et al.Effect of the PTEN gene on adhesion,invasion and metastasis of osteosarcoma cells[J].Oncol Rep,2014,32(4):1741-1747.
[23]CHEN MW,WU XJ.SLC25A22 promotes proliferation and metastasis of osteosarcoma cells via the PTEN signaling pathway[J].Technol Cancer Res Trest,2018,17:1533033818811143.
[24]TIAN K,DI R,WANG L.MicroRNA-23a enhances migration and invasion through PTEN in osteosarcoma[J].Cancer Gene Ther,2015,22(7):351-359.
[25]金晓昇,安慧敏,黄智铭.生存素和PTEN在良恶性胃溃疡中的表达[J].温州医科大学学报,2017,47(6):456-458.