miR-509靶向Rac1调节人肝癌LM3细胞侵袭和迁移及裸鼠模型的存活
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摘要
目的:探究微小RNA-509(miR-509)对人肝癌细胞生长、侵袭、迁移及荷瘤裸鼠存活的作用及其作用机制。方法:将miR-509模拟物(miR-509 mimic)和pcDNA Ras相关的C3肉毒毒素底物1(pcDNA Ras-related C3 botulinum toxin substrate 1, pcRac1)转染人肝癌LM3细胞,Western blot检测Rac1的表达;萤光素酶报告基因实验证明miR-509和Rac1的靶向关系;Transwell实验检测细胞侵袭能力;划痕实验检测细胞迁移能力;采用皮下注射LM3细胞的方法建立肝癌裸鼠移植瘤模型,检测裸鼠存活率,Western blot检测肿瘤组织Rac1的表达。结果:miR-509 mimic能明显抑制LM3细胞Rac1的表达(P<0.05),pcRac1能明显减弱miR-509 mimic对Rac1表达的抑制作用(P<0.05);miR-509 mimic还能显著减弱Rac1野生质粒的萤光素酶活性(P<0.05);同时,miR-509 mimic可减少侵袭细胞数,抑制肝癌细胞迁移(P<0.05)。此外,miR-509过表达还能升高肝癌移植瘤模型小鼠存活率(P<0.05),降低肿瘤组织Rac1的蛋白表达水平(P<0.01)。结论:miR-509能通过靶向抑制Rac1的表达而抑制肝癌细胞侵袭和迁移,并促进肝癌模型小鼠存活。
AIM: To investigate the effect of microRNA-509(miR-509) on the growth, invasion and migration of human hepatocellular carcinoma(HCC) LM3 cells and survival of tumor-bearing nude mice. METHODS: LM3 cells were transferred with miR-509 mimic and pcDNA Ras-related C3 botulinum toxin substrate 1(pcRac1), and the expression of Rac1 was measured by Western blot. The relationship between miR-509 and Rac1 was determined by luciferase reporter assay. The invasion ability was determined by Transwell assay, and the migration ability was measured by wound healing assay. Xenograft model of HCC was established by subcutaneous injection with LM3 cells into nude mice. The survival rate of the mice were recorded and the protein level of Rac1 was determined by Western blot. RESULTS: miR-509 mimic inhibited the expression of Rac1 in the LM3 cells(P<0.05). pcRac1 attenuated the effect of miR-509 on Rac1. miR-509 also alleviated luciferase activity of wild Rac1(P<0.05). Meanwhile, miR-509 mimic decreased the number of invasive LM3 cells and inhibited the migration of LM3 cells(P<0.05). In addition, over-expression of miR-509 up-regulated survival rate of model mice and decreased the protein level of Rac1 in the tumor tissue(P<0.01). CONCLUSION: miR-509 inhibits the invasion and migration of HCC cells and promotes the survival of tumor-bearing nude mice through inhibiting the expression of Rac1.
AIM: To investigate the effect of microRNA-509(miR-509) on the growth, invasion and migration of human hepatocellular carcinoma(HCC) LM3 cells and survival of tumor-bearing nude mice. METHODS: LM3 cells were transferred with miR-509 mimic and pcDNA Ras-related C3 botulinum toxin substrate 1(pcRac1), and the expression of Rac1 was measured by Western blot. The relationship between miR-509 and Rac1 was determined by luciferase reporter assay. The invasion ability was determined by Transwell assay, and the migration ability was measured by wound healing assay. Xenograft model of HCC was established by subcutaneous injection with LM3 cells into nude mice. The survival rate of the mice were recorded and the protein level of Rac1 was determined by Western blot. RESULTS: miR-509 mimic inhibited the expression of Rac1 in the LM3 cells(P<0.05). pcRac1 attenuated the effect of miR-509 on Rac1. miR-509 also alleviated luciferase activity of wild Rac1(P<0.05). Meanwhile, miR-509 mimic decreased the number of invasive LM3 cells and inhibited the migration of LM3 cells(P<0.05). In addition, over-expression of miR-509 up-regulated survival rate of model mice and decreased the protein level of Rac1 in the tumor tissue(P<0.01). CONCLUSION: miR-509 inhibits the invasion and migration of HCC cells and promotes the survival of tumor-bearing nude mice through inhibiting the expression of Rac1.
引文
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[22] Wang Z,Pedersen E,Basse A,et al.Rac1 is crucial for Ras-dependent skin tumor formation by controlling Pak1-Mek-Erk hyperactivation and hyperproliferation in vivo[J].Oncogene,2010,29(23):3362-3373.
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[27] Zhang Y,Dai Q,Zeng F,et al.MALAT1 promotes the proliferation and metastasis of osteosarcoma cells by activating the Rac1/JNK Pathway via targeting miR-509[J].Oncol Res,2018 Apr 27.[Epub ahead of print]
[2] Dhanasekaran R,Limaye A,Cabrera R.Hepatocellular carcinoma:current trends in worldwide epidemiology,risk factors,diagnosis,and therapeutics[J].Hepat Med,2012,4:19-37.
[3] Chen W,Zheng R,Baade PD,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[4] Takayama T.Surgical treatment for hepatocellular carcinoma[J].JPN J Clin Oncol,2011,41(4):447-454.
[5] Bodzin AS,Busuttil RW.Hepatocellular carcinoma:Advances in diagnosis,management,and long term outcome[J].World J Hepatol,2015,7(9):1157-1167.
[6] 李春雨,王琪,申珅,等.冬凌草甲素对人肝癌MHCC-97H细胞系侵袭和迁移的作用[J].中国病理生理杂志,2017,33(8):1423-1427.
[7] Turchinovich A,Weiz L,Langheinz A,et al.Characte-rization of extracellular circulating microRNA[J].Nucleic Acids Res,2011,39(16):7223-7233.
[8] Di Leva G,Garofalo M,Croce CM.MicroRNAs in cancer[J].Annu Rev Pathol,2014,9:287-314.
[9] Friedman R,Farh KC,Bartel D.Most mammalian mRNAs are conserved targets of microRNAs[J].Genome Res,2009,19(1):92-105.
[10] Xing F,Sharma S,Liu Y,et al.miR-509 suppresses brain metastasis of breast cancer cells by modulating RhoC and TNF- α[J].Oncogene,2015,34(37):4890-4900.
[11] Pan Y,Robertson G,Pedersen L,et al.miR-509-3p is clinically significant and strongly attenuates cellular migration and multi-cellular spheroids in ovarian cancer[J].Oncotarget,2016,7(18):25930-25948.
[12] Wang XH,Lu Y,Liang JJ,et al.MiR-509-3-5p causes aberrant mitosis and anti-proliferative effect by suppression of PLK1 in human lung cancer A549 cells[J].Biochem Biophys Res Commun,2016,478(2):676-682.
[13] Ren ZJ,Nong XY,Lv YR,et al.Mir-509-5p joins the Mdm2/p53 feedback loop and regulates cancer cell growth[J].Cell Death Dis,2014,5(8):e1387.
[14] Hammond SM.An overview of microRNAs[J].Adv Drug Deliv Rev,2015,87:3-14.
[15] Shukla K,Sharma AK,Ward A,et al.MicroRNA-30c-2-3p negatively regulates NF-κB signaling and cell cycle progression through downregulation of TRADD and CCNE1 in breast cancer[J].Mol Oncol,2015,9(6):1106-1119.
[16] Wang X,Qiu W,Zhang G,et al.MicroRNA-204 targets JAK2 in breast cancer and induces cell apoptosis through the STAT3/BCl-2/survivin pathway[J].Int J Clin Exp Pathol,2015,8(5):5017-5025.
[17] Ma F,Zhang J,Zhong L,et al.Upregulated microRNA-301a in breast cancer promotes tumor metastasis by targeting PTEN and activating Wnt/β-catenin signaling [J].Gene,2014,535(2):191-197.
[18] Geng L,Chaudhuri A,Talmon G,et al.MicroRNA-192 suppresses liver metastasis of colon cancer[J].Oncogene,2014,33(46):5332-5340.
[19] Zhang Y,Lin C,Liao G,et al.MicroRNA-506 suppresses tumor proliferation and metastasis in colon cancer by directly targeting the oncogene EZH2[J].Oncotarget,2015,6(32):32586-32601.
[20] Chen W,Du J,Li X,et al.miR-509-3p promotes cisplatin-induced apoptosis in ovarian cancer cells through the regulation of anti-apoptotic genes[J].Pharmacogenomics,2017,18(18):1671-1682.
[21] Zhang J,Zhu Z,Sheng J,et al.miR-509-3-5P inhibits the invasion and lymphatic metastasis by targeting PODXL and serves as a novel prognostic indicator for gastric cancer[J].Oncotarget,2017,8(21):34867-34883.
[22] Wang Z,Pedersen E,Basse A,et al.Rac1 is crucial for Ras-dependent skin tumor formation by controlling Pak1-Mek-Erk hyperactivation and hyperproliferation in vivo[J].Oncogene,2010,29(23):3362-3373.
[23] Bid HK,Roberts RD,Manchanda PK,et al.RAC1:an emerging therapeutic option for targeting cancer angiogenesis and metastasis[J].Mol Cancer Ther,2013,12(10):1925-1934.
[24] Chang JS,Su CY,Yu WH,et al.GIT1 promotes lung cancer cell metastasis through modulating Rac1/Cdc42 activity and is associated with poor prognosis[J].Oncotarget,2015,6(34):36278-36291.
[25] Zhu X,Gao G,Chu K,et al.Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer[J].Int J Biochem Cell Biol,2015,61:103-114.
[26] Yoon S,Han E,Choi YC,et al.Inhibition of cell proli-feration and migration by miR-509-3p that targets CDK2,Rac1,and PIK3C2A[J].Mol Cell,2014,37(4):314-321.
[27] Zhang Y,Dai Q,Zeng F,et al.MALAT1 promotes the proliferation and metastasis of osteosarcoma cells by activating the Rac1/JNK Pathway via targeting miR-509[J].Oncol Res,2018 Apr 27.[Epub ahead of print]