Mirtron类microRNA6894-5p过表达促进胃癌细胞的迁移、侵袭和增殖
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摘要
该文主要探讨Mirtron类micro RNA6894-5p过表达对胃癌细胞迁移、侵袭和增殖的影响。将miRNA6894-5p的模拟物分别转染进入胃癌MGC803和SGC7901细胞中,构建miRNA6894-5p过表达的细胞系;实时荧光定量PCR测miRNA6894-5p的RNA表达;Transwell实验检测细胞迁移侵袭能力;Cell Counting Kit 8实验检测细胞的增殖能力;荧光素酶报告基因实验检测miRNA6894-5p与肝配蛋白A3的靶向关系;实时荧光定量PCR和蛋白质印迹法进一步检测miRNA6894-5p过表达后EFNA3的m RNA和蛋白的变化。结果显示,成功构建miRNA6894-5p过表达模型的胃癌细胞系;与相应阴性对照组相比,过表达miRNA6894-5p可提高细胞迁移侵袭能力(P<0.05)和增强细胞增殖能力(P<0.05);荧光素酶报告基因实验证实,miRNA6894-5p靶向作用于肝配蛋白A3,过表达miRNA6894-5p后肝配蛋白A3的m RNA及蛋白水平显著下降(P<0.05)。该研究结果显示,Mirtron类miRNA6894-5p在人胃癌细胞中过表达可促进胃癌细胞的迁移、侵袭和增殖。
This article was to investigate the effects of human micro RNA6894-5 p(miRNA6894-5 p) on migration, invasion and proliferation of human gastric carcinoma cells. Mimics of miRNA6894-5 p were constructed and transfected into gastric cancer cells. The expression of miRNA6894-5 p in gastric carcinoma cells after transfection was determined by quantitative real time PCR(q RT-PCR). The migration, invasion and proliferation ability of gastric cancer cells were detected by Transwell chamber test and Cell Counting Kit-8 assays respectively. Luciferase reporter gene assay was used to detect the targeted relationship of miRNA6894-5 p and hepatic protein A3. The m RNA and protein expression of hepatic protein A3 were detected by q RT-PCR and Western blot. The results showed that micro RNA6894-5 p mimics were successfully transfected into gastric carcinoma cells. Compared with control cells, the ability of migration and invasion were significantly increased in overexpression cells(P<0.05) and miRNA6894-5 p overexpression could significantly promote gastric cancercells proliferation(P<0.05). The relationship between miRNA6894-5 p and EFNA3 was confirmed by Luciferase reporter gene assay. The m RNA and protein expression of EFNA3 were downregulated in miRNA6894-5 p overexpression cell. The above results indicate that miRNA6894-5 p can promote the migration, invasion and proliferation of gastric cancer cells.
This article was to investigate the effects of human micro RNA6894-5 p(miRNA6894-5 p) on migration, invasion and proliferation of human gastric carcinoma cells. Mimics of miRNA6894-5 p were constructed and transfected into gastric cancer cells. The expression of miRNA6894-5 p in gastric carcinoma cells after transfection was determined by quantitative real time PCR(q RT-PCR). The migration, invasion and proliferation ability of gastric cancer cells were detected by Transwell chamber test and Cell Counting Kit-8 assays respectively. Luciferase reporter gene assay was used to detect the targeted relationship of miRNA6894-5 p and hepatic protein A3. The m RNA and protein expression of hepatic protein A3 were detected by q RT-PCR and Western blot. The results showed that micro RNA6894-5 p mimics were successfully transfected into gastric carcinoma cells. Compared with control cells, the ability of migration and invasion were significantly increased in overexpression cells(P<0.05) and miRNA6894-5 p overexpression could significantly promote gastric cancercells proliferation(P<0.05). The relationship between miRNA6894-5 p and EFNA3 was confirmed by Luciferase reporter gene assay. The m RNA and protein expression of EFNA3 were downregulated in miRNA6894-5 p overexpression cell. The above results indicate that miRNA6894-5 p can promote the migration, invasion and proliferation of gastric cancer cells.
引文
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4 Wilk G,Braun R.Integrative analysis reveals disrupted pathways regulated by microRNAs in cancer.Nucleic Acids Res 2018;46(3):1089-101.
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7 Kim VN.MicroRNA biogenesis:coordinated cropping and dicing.Nat Rev Mol Cell Biol 2005;6(5):376-85.
8 Berezikov E,Chung WJ,Willis J,Cuppen E,Lai EC.Mammalian mirtron genes.Mol Cell 2007;28(2):328-36.
9 Sakai E,Miura Y,Suzuki-Kouyama E,Oka K,Tachibana M,Kawabata K,et al.A mammalian mirtron miR-1224 promotes tube-formation of human primary endothelial cells by targeting anti-angiogenic factor epsin2.Sci Rep 2017;7(1):5541.
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13 Ladewig E,Okamura K,Flynt AS,Westholm JO,Lai EC.Discovery of hundreds of mirtrons in mouse and human small RNA data.Genome Res 2012;22(9):1634-45.
14 Zhang H,Hou Y,Xu L,Zeng Z,Wen S,Du YE,et al.Cytoplasmic Drosha is aberrant in precancerous lesions of gastric carcinoma and its loss predicts worse outcome for gastric cancer patients.Dig Dis Sci 2016;61(4):1080-90.
15 Xu L,Hou Y,Tu G,Chen Y,Du YE,Zhang H,et al.Nuclear Drosha enhances cell invasion via an EGFR-ERK1/2-MMP7signaling pathway induced by dysregulated miRNA-622/197 and their targets LAMC2 and CD82 in gastric cancer.Cell Death Dis2017;8(3):e2642.
16石晓磊.MicroRNA-148a/b在胃癌中的表达及其功能与机制研究.第四军医大学(Xiaolei Shi.The expression and functional study of microRNA-148a/b in gastric cancer.Fourth Medical University),2011.
17赵晓迪.microRNA-7调控胃癌恶性生物学行为的功能与分子机制研究.第四军医大学(Xiaodi Zhao.Functional elucidation&mechanistic study of microRNA-7 in gastric carcinogenesis.Fourth Medical University),2015.
18 Shukla A,Dahiya S,Onteru SK,Singh D.Differentially expressed miRNA-210 during follicular-luteal transition regulates pre-ovulatory granulosa cell function targeting HRas and EFNA3.J Cell Biochem 2017;doi:10.1002/jcb.26508.
19 Wang Z,Liu Z,Liu B,Liu G,Wu S.Dissecting the roles of Ephrin-A3 in malignant peripheral nerve sheath tumor by TALENs.Oncol Rep 2015;34(1):391-8.
20 Wang Z,Yin B,Wang B,Ma Z,Liu W,Lv G.MicroRNA-210promotes proliferation and invasion of peripheral nerve sheath tumor cells targeting EFNA3.Oncol Res 2013;21(3):145-54.
2 Gong J,Li J,Wang Y,Liu C,Jia H,Jiang C,et al.Characterization of microRNA-29 family expression and investigation of their mechanistic roles in gastric cancer.Carcinogenesis 2014;35(2):497-506.
3 Imamura T,Komatsu S,Ichikawa D,Miyamae M,Okajima W,Ohashi T,et al.Low plasma levels of miR-101 are associated with tumor progression in gastric cancer.Oncotarget 2017;8(63):106538-50.
4 Wilk G,Braun R.Integrative analysis reveals disrupted pathways regulated by microRNAs in cancer.Nucleic Acids Res 2018;46(3):1089-101.
5 Watkin RL,Fitzpatrick GG,Kerrigan SW.The evolving role of MicroRNAs in endothelial cell dysfunction in response to infection.Semin Thromb Hemost 2018;44(3):216-23.
6 Koga T,Migita K,Sato T,Sato S,Umeda M,Nonaka F,et al.MicroRNA-204-3p inhibits lipopolysaccharide-induced cytokines in familial Mediterranean fever via the phosphoinositide 3-kinase gamma pathway.Rheumatology(Oxford,England)2017;57(4):718-26.
7 Kim VN.MicroRNA biogenesis:coordinated cropping and dicing.Nat Rev Mol Cell Biol 2005;6(5):376-85.
8 Berezikov E,Chung WJ,Willis J,Cuppen E,Lai EC.Mammalian mirtron genes.Mol Cell 2007;28(2):328-36.
9 Sakai E,Miura Y,Suzuki-Kouyama E,Oka K,Tachibana M,Kawabata K,et al.A mammalian mirtron miR-1224 promotes tube-formation of human primary endothelial cells by targeting anti-angiogenic factor epsin2.Sci Rep 2017;7(1):5541.
10 Butkyte S,Ciupas L,Jakubauskiene E,Vilys L,Mocevicius P,Kanopka A,et al.Splicing-dependent expression of microRNAs of mirtron origin in human digestive and excretory system cancer cells.Clin Epigenetics 2016;8:33.
11 Kim YK,Kim B,Kim VN.Re-evaluation of the roles of DROSHA,Export in 5,and DICER in microRNA biogenesis.Proc Natl Acad Sci USA 2016;113(13):E1881-9.
12 Flynt AS,Greimann JC,Chung WJ,Lima CD,Lai EC.MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila.Mol Cell 2010;38(6):900-7.
13 Ladewig E,Okamura K,Flynt AS,Westholm JO,Lai EC.Discovery of hundreds of mirtrons in mouse and human small RNA data.Genome Res 2012;22(9):1634-45.
14 Zhang H,Hou Y,Xu L,Zeng Z,Wen S,Du YE,et al.Cytoplasmic Drosha is aberrant in precancerous lesions of gastric carcinoma and its loss predicts worse outcome for gastric cancer patients.Dig Dis Sci 2016;61(4):1080-90.
15 Xu L,Hou Y,Tu G,Chen Y,Du YE,Zhang H,et al.Nuclear Drosha enhances cell invasion via an EGFR-ERK1/2-MMP7signaling pathway induced by dysregulated miRNA-622/197 and their targets LAMC2 and CD82 in gastric cancer.Cell Death Dis2017;8(3):e2642.
16石晓磊.MicroRNA-148a/b在胃癌中的表达及其功能与机制研究.第四军医大学(Xiaolei Shi.The expression and functional study of microRNA-148a/b in gastric cancer.Fourth Medical University),2011.
17赵晓迪.microRNA-7调控胃癌恶性生物学行为的功能与分子机制研究.第四军医大学(Xiaodi Zhao.Functional elucidation&mechanistic study of microRNA-7 in gastric carcinogenesis.Fourth Medical University),2015.
18 Shukla A,Dahiya S,Onteru SK,Singh D.Differentially expressed miRNA-210 during follicular-luteal transition regulates pre-ovulatory granulosa cell function targeting HRas and EFNA3.J Cell Biochem 2017;doi:10.1002/jcb.26508.
19 Wang Z,Liu Z,Liu B,Liu G,Wu S.Dissecting the roles of Ephrin-A3 in malignant peripheral nerve sheath tumor by TALENs.Oncol Rep 2015;34(1):391-8.
20 Wang Z,Yin B,Wang B,Ma Z,Liu W,Lv G.MicroRNA-210promotes proliferation and invasion of peripheral nerve sheath tumor cells targeting EFNA3.Oncol Res 2013;21(3):145-54.