microRNA-31通过靶向调控Dock1抑制乳腺癌的上皮-间质转化
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摘要
目的:探讨microRNA-31(miR-31)靶向调控Dock1对乳腺癌上皮-间质转化的影响。方法:通过qRT-PCR、Western blot检测人正常乳腺上皮细胞与乳腺癌细胞中miR-31、Dock1的表达情况;将含有miR-31的过表达质粒或Dock1的敲除质粒转入乳腺癌细胞MDA-MB-231中并检测转染效率;用Western blot检测转染后各组细胞中Dock1的表达变化; Transwell侵袭实验检测转染及共转染后各组细胞的侵袭能力的变化; Western blot检测转染及共转染后各组细胞中EMT标志物的表达情况。结果:MCF-7细胞中miR-31的表达明显高于MDA-MB-231细胞,但两组都低于其在MCF-10A中的表达。转染对照及过表达质粒后,MDA-MB-231细胞中miR-31的表达较正常组与对照组明显上调,Dock1表达明显下调。Transwell侵袭实验结果显示,MDA-MB-231/miR-31组相比于MDA-MB-231/NC组穿过基底膜的细胞数明显减少,而与MDA-MB-231/miR-31+con组相比无明显差距; MDA-MB-231/miR-31+Dock1组与MDA-MB-231/miR-31+con组相比穿过基底膜的细胞数明显增多。Western blot结果显示,MDA-MB-231/miR-31和MDA-MB-231/miR-31+con细胞相比于MDA-MB-231/NC细胞中E-cadherin表达水平显著上调,在MDA-MB-231/miR-31+Dock1细胞中E-cadherin的表达水平与对照组相比无统计学意义;与MDA-MB-231/NC细胞相比,MDA-MB-231/miR-31和MDA-MB-231/miR-31+con细胞中Vimentin表达水平明显下调,MDA-MB-231/miR-31+Dock1细胞中Vimentin的表达水平几乎无变化。结论:miR-31可以靶向调控Dock1来抑制乳腺癌的上皮-间质转化,进而抑制乳腺癌的侵袭与转移。
Objective: To investigate the effect of microRNA-31( miR-31) in breast cancer of epithelial-mesenchymal transition by targeting Dock1. Methods: The expression of miR-31 and Dock1 in human normal mammary epithelial cells and breast cancer cells were detected by qRT-PCR and Western blot. The overexpression plasmids of miR-31 or the knock-out plasmids of Dock1 was transfected into the breast cancer cells MDA-MB-231 and the transfection efficiency was detected. Western blot was used to detect the expression of Dock1 in each group after transfection. Transwell invasion assay was used to detect the invasiveness of cells in each group after transfection or cotransfection. Western blot was used to detect the expression of EMT markers in transfected cells or cotransfected cells. Results: The expression of miR-31 in MCF-7 cells was significantly higher than MDA-MB-231 cells,but the expression of the two groups was lower than MCF-10 A cells. After transfection of control and overexpression plasmids,the expression of miR-31 in MDA-MB-231 cells was significantly higher than the normal group and the control group,and the expression of Dock1 was obviously down regulated. Transwell invasion assay showed that the number of cells in MDA-MB-231/miR-31 group compared with MDA-MB-231/NC group through the basement membrane was significantly reduced,but no significant difference compared with the MDA-MB-231/miR-31 +con group,MDA-MB-231/miR-31+Dock1 group compared with MDA-MB-231/miR-31+con group,the number of cells through the basement membrane increased significantly. Western blot results showed that MDA-MB-231/miR-31 and MDA-MB-231/miR-31 + con cells compared with MDA-MB-231/NC cells the expression level of E-cadherin were significant increased,the E-cadherin expression level in MDA-MB-231/miR-31+Dock1 cells compared with the control group was not statistically significant. Comparing with MDA-MB-231/NC cells,the level of Vimentin was significantly down-regulated,the expression of Vimentin in MDA-MB-231/miR-31+Dock1 cells no change almost. Conclusion: miR-31 can inhibits the epithelial-mesenchymal transition of breast cancer by targeting Dock1,and then inhibits the invasion and metastasis of breast cancer.
Objective: To investigate the effect of microRNA-31( miR-31) in breast cancer of epithelial-mesenchymal transition by targeting Dock1. Methods: The expression of miR-31 and Dock1 in human normal mammary epithelial cells and breast cancer cells were detected by qRT-PCR and Western blot. The overexpression plasmids of miR-31 or the knock-out plasmids of Dock1 was transfected into the breast cancer cells MDA-MB-231 and the transfection efficiency was detected. Western blot was used to detect the expression of Dock1 in each group after transfection. Transwell invasion assay was used to detect the invasiveness of cells in each group after transfection or cotransfection. Western blot was used to detect the expression of EMT markers in transfected cells or cotransfected cells. Results: The expression of miR-31 in MCF-7 cells was significantly higher than MDA-MB-231 cells,but the expression of the two groups was lower than MCF-10 A cells. After transfection of control and overexpression plasmids,the expression of miR-31 in MDA-MB-231 cells was significantly higher than the normal group and the control group,and the expression of Dock1 was obviously down regulated. Transwell invasion assay showed that the number of cells in MDA-MB-231/miR-31 group compared with MDA-MB-231/NC group through the basement membrane was significantly reduced,but no significant difference compared with the MDA-MB-231/miR-31 +con group,MDA-MB-231/miR-31+Dock1 group compared with MDA-MB-231/miR-31+con group,the number of cells through the basement membrane increased significantly. Western blot results showed that MDA-MB-231/miR-31 and MDA-MB-231/miR-31 + con cells compared with MDA-MB-231/NC cells the expression level of E-cadherin were significant increased,the E-cadherin expression level in MDA-MB-231/miR-31+Dock1 cells compared with the control group was not statistically significant. Comparing with MDA-MB-231/NC cells,the level of Vimentin was significantly down-regulated,the expression of Vimentin in MDA-MB-231/miR-31+Dock1 cells no change almost. Conclusion: miR-31 can inhibits the epithelial-mesenchymal transition of breast cancer by targeting Dock1,and then inhibits the invasion and metastasis of breast cancer.
引文
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[2]Lee J,Park S,Kim S,et al.Characteristics and survival of breast cancer patients with multiple synchronous or metachronous primary cancers[J].Yonsei Med J,2015,56(5):1213-1220.
[3]Qiu Y,Liu Y,Li WH,et al.P2Y2 receptor promotes the migration and invasion of breast cancer cells via EMT-related genes Snail and E-cadherin[J].Oncol Rep,2018,39(1):138-150.
[4]Muti P,Donzelli S,Sacconi A,et al.MiRNA-513a-5p inhibits progesterone receptor expression and constitutes a risk factor for breast cancer:the ORDET prospective study[J].Carcinogenesis,2018,39(2):98-108.
[5]Zhao X,Wang Y,Deng R,et al.miR186 suppresses prostate cancer progression by targeting Twist1[J].Oncotarget,2016,7(22):33136-33151.
[6]Ma F,Zhang L,Ma L,et al.MiR-361-5p inhibits glycolytic metabolism,proliferation and invasion of breast cancer by targeting FGFR1 and MMP-1[J].J Exp Clin Cancer Res,2017,36(1):158.
[7]Dalmay T.Mechanism of miRNA-mediated repression of mRNAtranslation[J].Essays Biochem,2013,54:29-38.
[8]Kim HS,Lee KS,Bae HJ,et al.MicroRNA-31 functions as a tumor suppressor by regulating cell cycle and epithelial-mesenchymal transition regulatory proteins in liver cancer[J].Oncotarget,2015,6(10):8089-8102.
[9]Yu M,Liang H,Fu Z,et al.BAP1 suppresses lung cancer progression and is inhibited by miR-31[J].Oncotarget,2016,7(12):13742-13753.
[10]Ge F,Wang C,Wang W,et al.MicroRNA-31 inhibits tumor invasion and metastasis by targeting Rho A in human gastric cancer[J].Oncol Rep,2017,38(2):1133-1139.
[11]Pan J,Lu F,Xu H,et al.Low p21 level is necessary for the suppressive effects of micoRNA-31 on glioma cell migration and invasion[J].Tumour Biol,2016,37(7):9663-9670.
[12]Wang H,Liu W,Black S,et al.Kaiso,a transcriptional repressor,promotes cell migration and invasion of prostate cancer cells through regulation of miR-31 expression[J].Oncotarget,2016,7(5):5677-5689.
[13]Luo LJ,Yang F,Ding J,et al.MiR-31 inhibits migration and invasion by targeting SATB2 in triple negative breast cancer[J].Gene,2016,594(1):47-58.
[14]Zhang B,Li H,Yin C,et al.Dock1 promotes the mesenchymal transition of glioma and is modulated by MiR-31[J].Neuropathol Appl Neurobiol,2017,43(5):419-32.
[15]Sun L,Zhang B,Liu Y,et al.MiR125a-5p acting as a novel Gab2suppressor inhibits invasion of glioma[J].Mol Carcinog,2016,55(1):40-51.
[16]Li H,Yin C,Zhang B,et al.PTTG1 promotes migration and invasion of human non-small cell lung cancer cells and is modulated by miR-186[J].Carcinogenesis,2013,34(9):2145-2155.
[17]Shi L,Wang Y,Lu Z,et al.miR-127 promotes EMT and stem-like traits in lung cancer through a feed-forward regulatory loop[J].Oncogene,2017,36(12):1631-1643.
[18]Barber AG,Castillo-Martin M,Bonal DM,et al.PI3K/AKTpathway regulates E-cadherin and Desmoglein 2 in aggressive prostate cancer[J].Cancer Med,2015,4(8):1258-1271.
[19]Stepicheva NA,Song JL.Function and regulation of microRNA-31in development and disease[J].Mol Reprod Dev,2016,83(8):654-674.
[20]Gao Y,Yi J,Zhang K,et al.Downregulation of MiR-31 stimulates expression of LATS2 via the hippo pathway and promotes epithelial-mesenchymal transition in esophageal squamous cell carcinoma[J].J Exp Clin Cancer Res,2017,36(1):161.
[21]Cottonham CL,Kaneko S,Xu L.miR-21 and miR-31 converge on TIAM1 to regulate migration and invasion of colon carcinoma cells[J].J Biol Chem,2010,285(46):35293-35302.
[22]Chen DJ,Chen W,Jiang H,et al.Downregulation of DOCK1sensitizes bladder cancer cells to cisplatin through preventing epithelial-mesenchymal transition[J].Drug Des Devel Ther,2016,10:2845-2853.
[23]Wang H,Linghu H,Wang J,et al.The role of Crk/Dock180/Rac1 pathway in the malignant behavior of human ovarian cancer cell SKOV3[J].Tumour Biol,2010,31(1):59-67.
[24]Li H,Yang L,Fu H,et al.Association between Galphai2 and ELMO1/Dock180 connects chemokine signalling with Rac activation and metastasis[J].Nat Commun,2013,4:1706.
[25]Shi L,Zhang B,Sun X,et al.CC chemokine ligand 18(CCL18)promotes migration and invasion of lung cancer cells by binding to Nir1 through Nir1-ELMO1/DOC180 signaling pathway[J].Mol Carcinog,2016,55(12):2051-2062.