miRNA-138模拟物转染对人乳腺癌细胞迁移、侵袭能力的影响及其机制
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摘要
目的观察miRNA-138模拟物(miRNA-138 mimics)对人乳腺癌细胞迁移、侵袭能力的影响,并探讨其可能机制。方法选取人高侵袭性乳腺细胞系(MDA-MB-231、MDA-MB-468)及人低侵袭性乳腺癌细胞系(MCF-7)和人正常乳腺上皮细胞系(HBL-100),qRT-PCR法检测微小RNA-138(miRNA-138);取MDA-MB-231并分为两组,miRNA-138 mimics组细胞系以miRNA-138 mimics转染,miR-NC组细胞系以miR阴性对照模拟物(miR-NC)转染,qRT-PCR法检测miRNA-138,Transwell迁移和侵袭实验测算迁移细胞数、侵袭细胞数,Western blotting法检测SOX4蛋白及上皮间质转化(EMT)相关标志物(E-cadherin、Fibronectin、Vimentin蛋白)。结果 MDA-MB-231、MDA-MB-468、MCF-7、HBL-100中miRNA-138相对表达量分别为0.23±0.08、0.42±0.05、0.56±0.11、1.03±0.04,MDAMB-231、MDA-MB-468、MCF-7与HBL-100比较,MDA-MB-231、MDA-MB-468与MCF-7比较,P均<0.05。miRNA-138 mimics组和miR-NC组miRNA-138相对表达量分别为13.35±0.35、0.98±0.05,两组比较,P <0.05。miRNA-138 mimics组和miR-NC组迁移细胞数分别为(210±30)、(340±18)个,侵袭细胞数分别为(100±16)、(190±26)个,两组比较,P均<0.05。miRNA-138 mimics组SOX、E-cadherin、Fibronectin、Vimentin蛋白灰度值分别为0.16±0.08、0.59±0.16、0.42±0.14、0.17±0.09,miR-NC组分别为0.77±0.19、0.12±0.06、1.19±0.21、0.44±0.12,两组比较,P均<0.05。结论 miRNA-138 mimics能够抑制乳腺癌细胞侵袭和迁移,机制可能与其调控SOX4基因抑制乳腺癌细胞EMT有关。
Objective To observe the effects of miRNA-138 mimics on the migration and invasion of human breast cancer cells,and to explore its possible mechanism.Methods Human highly invasive breast cell lines(MDA-MB-231 and MDA-MB-468),human low invasive breast cancer cell lines(MCF-7) and human normal breast epithelial cell line(HBL-100) were selected.The quantitative real-time fluorescence PCR(qRT-PCR) was used to detect the expression of miRNA-138 in different cell lines.MDA-MB-231 cells were selected and divided into two groups.The cells in the miRNA-138 mimics group were transfected with miRNA-138 mimics,and the miR-NC group with miRNA negative control(miR-NC).The expression of miRNA-138 was detected by qRT-PCR,the number of migrating cells and invasive cells was measured by Transwell migration and invasion tests,and the expression levels of SOX4,E-cadherin,Fibronectin,and Vimentin proteins were detected by Western blotting.Results The relative expression levels of miRNA-138 in the MDA-MB-231,MDA-MB-468,MCF-7 and HBL-100 cells were 0.23±0.08,0.42±0.05,0.56±0.11,and 1.03±0.04,respectively;significant difference was found between MDA-MB-231,MDA-MB-468,MCF-7 cells and HBL-100 cells,and between MDA-MB-231,MDA-MB-468 cells and MCF-7 cells,(all P<0.05).The relative expression levels of miRNA-138 in the miRNA-138 mimics group and the miR-NC group were 13.35±0.35 and 0.98±0.05,respectively,with significant difference(P<0.05).The number of migrating cells in the miRNA-138 mimics group and miR-NC group were 210±30 and 340±18,respectively,and the invasive cells were 100±16 and 190±26,respectively,with significant difference(both P<0.05).The gray values of SOX,E-cadherin,Fibronectin,and Vimentin proteins in the miRNA-138 mimics group were 0.16±0.08,0.59±0.16,0.42±0.14,and 0.17±0.09,which were 0.77±0.19,0.12±0.06,1.19±0.21,and 0.44±0.12,respectively,in the miR-NC group,with significant difference(all P<0.05).Conclusion The miRNA-138 mimics can inhibit the invasion and migration of breast cancer cells,and its mechanism may be related to the inhibition of EMT of breast cancer cells by regulation SOX4 gene.
Objective To observe the effects of miRNA-138 mimics on the migration and invasion of human breast cancer cells,and to explore its possible mechanism.Methods Human highly invasive breast cell lines(MDA-MB-231 and MDA-MB-468),human low invasive breast cancer cell lines(MCF-7) and human normal breast epithelial cell line(HBL-100) were selected.The quantitative real-time fluorescence PCR(qRT-PCR) was used to detect the expression of miRNA-138 in different cell lines.MDA-MB-231 cells were selected and divided into two groups.The cells in the miRNA-138 mimics group were transfected with miRNA-138 mimics,and the miR-NC group with miRNA negative control(miR-NC).The expression of miRNA-138 was detected by qRT-PCR,the number of migrating cells and invasive cells was measured by Transwell migration and invasion tests,and the expression levels of SOX4,E-cadherin,Fibronectin,and Vimentin proteins were detected by Western blotting.Results The relative expression levels of miRNA-138 in the MDA-MB-231,MDA-MB-468,MCF-7 and HBL-100 cells were 0.23±0.08,0.42±0.05,0.56±0.11,and 1.03±0.04,respectively;significant difference was found between MDA-MB-231,MDA-MB-468,MCF-7 cells and HBL-100 cells,and between MDA-MB-231,MDA-MB-468 cells and MCF-7 cells,(all P<0.05).The relative expression levels of miRNA-138 in the miRNA-138 mimics group and the miR-NC group were 13.35±0.35 and 0.98±0.05,respectively,with significant difference(P<0.05).The number of migrating cells in the miRNA-138 mimics group and miR-NC group were 210±30 and 340±18,respectively,and the invasive cells were 100±16 and 190±26,respectively,with significant difference(both P<0.05).The gray values of SOX,E-cadherin,Fibronectin,and Vimentin proteins in the miRNA-138 mimics group were 0.16±0.08,0.59±0.16,0.42±0.14,and 0.17±0.09,which were 0.77±0.19,0.12±0.06,1.19±0.21,and 0.44±0.12,respectively,in the miR-NC group,with significant difference(all P<0.05).Conclusion The miRNA-138 mimics can inhibit the invasion and migration of breast cancer cells,and its mechanism may be related to the inhibition of EMT of breast cancer cells by regulation SOX4 gene.
引文
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[16]Zhao M,Ang L,Huang J,et al.MicroRNAs regulate the epithelial-mesenchymal transition and influence breast cancer invasion and metastasis[J].Tumour Biol,2017,39(2):1010428317691682.
[17]Liu F,Wu L,Wang A,et al.MicroRNA-138 attenuates epithelial-to-mesenchymal transition by targeting SOX4 in clear cell renal cell carcinoma[J].Am J Transl Res,2017,9(8):3611-3622.
[18]Cai Q,Wang Z,Wang S,et al.Long non-coding RNA LINC00152promotes gallbladder cancer metastasis and epithelial-mesenchymal transition by regulating HIF-1αvia miR-138[J].Open Biol,2017,7(1):160247.
[19]Tiwari N,Tiwari VK,Waldmeier L,et al.Sox4 is a master regulator of epithelial-mesenchymal transition by controlling Ezh2 expression and epigenetic reprogramming[J].Cancer Cell,2013,23(6):768-783.
[2]程鸣佳,储沨婷,沈刚.miR-138与肿瘤的研究进展[J].肿瘤,2017,37(12):1334-1338.
[3]Chan SH,Huang WC,Chang JW,et al.MicroRNA-149 targets GIT1 to suppress integrin signaling and breast cancer metastasis[J].Oncogene,2014,33(36):4496-4507.
[4]Allemani C,Weir HK,Carreira H,et al.Global surveillance of cancer survival 1995-2009:analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries(CONCORD-2)[J].Lancet,2015,385(9972):977-1010.
[5]Hammond SM.An overview of microRNAs[J].Adv Drug DelivRev,2015,87:3-14.
[6]Erdmann K,Kaulke K,Rieger C,et al.MiR-26a and miR-138block the G1/S transition by targeting the cell cycle regulating network in prostate cancer cells[J].J Cancer Res Clin Oncol,2016,142(11):2249-2261.
[7]Han LP,Fu T,Lin Y,et al.MicroRNA-138 negatively regulates non-small cell lung cancer cells through the interaction with cyclin D3[J].Tumour Biol,2016,37(1):291-298.
[8]Zheng S,Zhang X,Wang X,et al.Downregulation of miR-138predicts poor prognosis in patients with esophageal squamous cell carcinoma[J].Cancer Biomark,2017,20(1):49-54.
[9]Li H,Sheng Y,Zhang Y,et al.MicroRNA-138 is a potential biomarker and tumor suppressor in human cervical carcinoma by reversely correlated with TCF3 gene[J].Gynecol Oncol,2017,145(3):569-576.
[10]Mittal V.Epithelial mesenchymal transition in aggressive lung cancers[J].Adv Exp Med Biol,2016,890:37-56.
[11]Yeung KT,Yang J.Epithelial-mesenchymal transition in tumor metastasis[J].Mol Oncol,2017,11(1):28-39.
[12]Lamouille S,Xu J,Derynck R.Molecular mechanisms of epithelial-mesenchymal transition[J].Nat Rev Mol Cell Biol,2014,15(3):178-196.
[13]Yeung KT,Yang J.Epithelial-mesenchymal transition in tumor metastasis[J].Mol Oncol,2017,11(1):28-39.
[14]Liao TT,Yang MH.Revisiting epithelial-mesenchymal transition in cancer metastasis:the connection between epithelial plasticity and stemness[J].Mol Oncol,2017,11(7):792-804.
[15]Lamouille S,Xu J,Derynck R.Molecular mechanisms of epithelial-mesenchymal transition[J].Nat Rev Mol Cell Biol,2014,15(3):178-196.
[16]Zhao M,Ang L,Huang J,et al.MicroRNAs regulate the epithelial-mesenchymal transition and influence breast cancer invasion and metastasis[J].Tumour Biol,2017,39(2):1010428317691682.
[17]Liu F,Wu L,Wang A,et al.MicroRNA-138 attenuates epithelial-to-mesenchymal transition by targeting SOX4 in clear cell renal cell carcinoma[J].Am J Transl Res,2017,9(8):3611-3622.
[18]Cai Q,Wang Z,Wang S,et al.Long non-coding RNA LINC00152promotes gallbladder cancer metastasis and epithelial-mesenchymal transition by regulating HIF-1αvia miR-138[J].Open Biol,2017,7(1):160247.
[19]Tiwari N,Tiwari VK,Waldmeier L,et al.Sox4 is a master regulator of epithelial-mesenchymal transition by controlling Ezh2 expression and epigenetic reprogramming[J].Cancer Cell,2013,23(6):768-783.