miR-302b-3p靶向SQSTM1调控乳腺癌MCF7细胞的自噬
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摘要
目的探讨miR-302b-3p靶向SQSTM1调控乳腺癌MCF7细胞的自噬。方法运用TargetScan在线分析miR-302b-3p与SQSTM1的相关性;将SQSTM1的3′UTR构建进PMIR-RB-REPORT质粒,利用luciferase assay检测miR-302b-3p是否靶向调控SQSTM1;用RNA转染试剂转染miR-302b-3p mimics或miR-302b-3p inhibitor进乳腺癌MCF7细胞,通过Western blot检测SQSTM1的表达量和乳腺癌MCF7细胞自噬标志蛋白表达情况。单丹磺酰尸胺染色检测细胞自噬发生水平。结果 miR-302b-3p靶向SQSTM1的3′UTR的584-590的位置;过表达miR-302b-3p时,SQSTM1的表达量明显下降(P<0.05),细胞自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ和Beclin1的表达量明显上升(P<0.05),MCF7自噬发生水平降低(P<0.05);敲低miR-302b-3p时,SQSTM1的表达量明显上升(P<0.05),细胞自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ和Beclin1的表达量明显下降(P<0.05),MCF7自噬发生水平升高(P<0.05)。结论 miR-302b-3p靶向SQSTM1的3′UTR后降低SQSTM1的蛋白水平,最终抑制乳腺癌细胞MCF7的自噬。
Objective To investigate whether miR-302 b-3 p targets SQSTM1 to regulate autophagy in breast cancer cells MCF7. Methods TargetScan was used to analyze the correlation between miR-302 b-3 p and SQSTM1;the 3′UTR of SQSTM1 was constructed into PMIR-RB-REPORT plasmid,and luciferase assay was used to detect whether miR-302 b-3 p targets SQSTM1;The staining reagent was transfected into miR-302 b-3 p mimics or miR-302 b-3 p inhibitor into breast cancer MCF7 cells. The expression of SQSTM1 and the expression of autophagy marker protein in breast cancer MCF7 cells were detected by western blot. Monodansylcadaverine staining was used to detect the level of autophagy. Results miR-302 b-3 p targeted the position of 584-590 of the 3′UTR of SQSTM1;when overexpressing miR-302 b-3 p,the expression of SQSTM1 was significantly decreased(P<0.05),autophagy-associated protein LC3-II The expression of/LC3-I and Beclin1 was significantly increased(P<0.05),and the level of autophagy was decreased in MCF7(P<0.05). The expression of SQSTM1 was significantly increased when knocking down miR-302 b-3 p(P<0.05). The expression levels of autophagy-related proteins LC3-II/LC3-I and Beclin1 were significantly decreased(P<0.05),and the levels of autophagy in MCF7 were increased(P<0.05). Conclusion miR-302 b-3 p targets the 3′UTR of SQSTM1 and decreases the protein level of SQSTM1,which ultimately inhibits autophagy in breast cancer cells MCF7.
Objective To investigate whether miR-302 b-3 p targets SQSTM1 to regulate autophagy in breast cancer cells MCF7. Methods TargetScan was used to analyze the correlation between miR-302 b-3 p and SQSTM1;the 3′UTR of SQSTM1 was constructed into PMIR-RB-REPORT plasmid,and luciferase assay was used to detect whether miR-302 b-3 p targets SQSTM1;The staining reagent was transfected into miR-302 b-3 p mimics or miR-302 b-3 p inhibitor into breast cancer MCF7 cells. The expression of SQSTM1 and the expression of autophagy marker protein in breast cancer MCF7 cells were detected by western blot. Monodansylcadaverine staining was used to detect the level of autophagy. Results miR-302 b-3 p targeted the position of 584-590 of the 3′UTR of SQSTM1;when overexpressing miR-302 b-3 p,the expression of SQSTM1 was significantly decreased(P<0.05),autophagy-associated protein LC3-II The expression of/LC3-I and Beclin1 was significantly increased(P<0.05),and the level of autophagy was decreased in MCF7(P<0.05). The expression of SQSTM1 was significantly increased when knocking down miR-302 b-3 p(P<0.05). The expression levels of autophagy-related proteins LC3-II/LC3-I and Beclin1 were significantly decreased(P<0.05),and the levels of autophagy in MCF7 were increased(P<0.05). Conclusion miR-302 b-3 p targets the 3′UTR of SQSTM1 and decreases the protein level of SQSTM1,which ultimately inhibits autophagy in breast cancer cells MCF7.
引文
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[2]王智,王维虎,王淑莲,等.基于聚类分析的不同表型pT1~2 N0期浸润性乳腺癌患者的预后差异[J].中华肿瘤杂志,2016,38(6):440-447.
[3]王丹丹,陈雪松.细胞自噬及其在乳腺癌中的研究进展[J].现代肿瘤医学,2017,25(22):3704-3707.
[4]纳米层状双氢氧化物共负载体系的制备及其抑制肝癌氟尿嘧啶耐药的协同效应机制研究[D].第二军医大学,2016.
[5] Thompson H G R,Harris J W,Wold B J,et al. p62overexpression in breast tumors and regulation by prostate-derived Ets factor in breast cancer cells[J].Oncogene,2003,22(15):2322.
[6] Guo B,Zhao Z,Wang Z,et al. MicroRNA-302b-3p Suppresses cell proliferation through AKT pathway by targeting IGF-1R in human gastric cancer[J]. Cellular Physiology and Biochemistry,2017:1701-1711.
[7] Li Q,Ran P,Zhang X,et al. Downregulation of Nacetylglucosaminyltransferase GCNT3 by miR-302b-3p decreases Non-Small Cell Lung Cancer(NSCLC)cell proliferation,migration and invasion[J]. Cellular Physiology and Biochemistry,2018,50(3):987-1004.
[8] Li Y,Huo J,Pan X,et al. Microrna 302b-3p/302c-3p/302d-3p inhibits epithelial-mesenchymal transition and promotes apoptosis in human endometrial carcinoma cells[J]. Onco Targets and therapy,2018,11:1275.
[9] Cataldo A,Cheung D G,Balsari A,et al. miR-302b enhances breast cancer cell sensitivity to cisplatin by regulating E2F1 and the cellular DNA damage response[J]. Oncotarget,2016,7(1):786.
[10]林佳,张晓晔,于莉,等.女性乳腺癌患者性激素与焦虑抑郁情绪的相关性研究[J].中国临床研究,2016,29(5):719-720.
[11]张科,梅金红,蔡勇,等. BRCA1、Ki67在不同分子分型乳腺癌中的表达及临床病理意义[J].实用癌症杂志,2016,31(10):1582-1586.
[12]张继博,史业辉,贾勇圣,等.三阴性乳腺癌治疗进展[J].肿瘤,2017,37(7):788-794.
[13]万华丽,张家玉.自噬在乳腺癌发生、发展中的机制和研究进展[J].海南医学,2017,28(1):119-121.
[14]伏静,孙晓溪.微小RNA在女性生殖调节中的研究进展[J].中华生殖与避孕杂志,2016,36(1):49-54.
[15]杜萌,朱宝生,吕涛. MicroRNAs对胎儿血红蛋白表达的调控作用[J].中国病理生理杂志,2017,33(5):956-960.
[16]Zhang L,Huang J,Yang N,et al. microRNAs exhibit high frequency genomic alterations in human cancer[J]. Proceedings of the National Academy of Sciences of the United States of America,2006,103(24):9136-9141.
[17]Chen D,Yang H. Integrated analysis of differentially expressed genes in breast cancer pathogenesis[J].Oncology Letters,2015,9(6):2560.
[18]韩颖,吴正升,吴强.乳腺癌新辅助化疗前后p62和XIAP表达的变化及其意义[J].临床与实验病理学杂志,2017,33(5):481-484.
[19]Shim H,Liang Z. MicroRNA-302 replacement therapy sensitizes breast cancer cells to ionizing radiation[J]. Pharmaceutical Research,2013,30(4):1008-1016.
[20]刘晓健,黄雷,陈睿琦,等.抑制自噬增强MCF-7乳腺癌细胞对依托泊苷的敏感性[J].基础医学与临床,2016,36(8):1087-1091.
[21]刘兆芸,贺科文,宋兴国,等.自噬抑制剂可增强三阴性乳腺癌细胞系MDA-MB-468和MDA-MB-231对吉非替尼的敏感性[J].中华肿瘤杂志,2016,38(6):417-424.
[22]刘兆芸.抑制自噬对三阴性乳腺癌EGFR靶向治疗的影响研究[D].济南大学,2017.
[23]万晓庆,王学健,王艺斐,等.循环microRNAs的外泌形成机制及其对乳腺癌侵袭和转移的影响[J].生物化学与生物物理进展,2017,44(4):269-278.
[24]张创杰.胰腺癌中自噬相关蛋白Beclin1、LC3和P62的表达及临床意义[D].郑州大学,2015.