Effect of miR-7 Overexpression on Biological Behavior of Colon Carcinoma HCT-116 Cells
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摘要:目的
探讨上调microRNA-7（miR-7）表达对结肠癌HCT-116细胞生物学行为的影响及可能机制。
方法HCT-116转染后分成miR-7 mimics组、Scramble（阴性对照）组和空白对照组。实时荧光定量PCR（RT-Fq-PCR）检测转染48 h后各组细胞中miR-7表达水平，CCK-8法检测转染72 h后各组细胞的增殖抑制情况，Transwell实验观察转染24 h后细胞侵袭能力，流式细胞仪行细胞周期和细胞凋亡检测。Western blot检测PI3K（p110）、p-Akt及p-mTOR的蛋白表达。
结果miR-7 mimics组miR-7表达水平明显上调，与其余两组比较差异有统计学意义（P < 0.05）；与阴性对照组和空白对照组相比，miR-7 mimics组细胞增殖抑制率升高，侵袭能力降低，细胞周期分析提示G0/G1期的细胞比例增高，S期细胞明显减少，细胞凋亡率明显增加，且PI3K、p-Akt及p-mTOR的蛋白水平明显降低（P < 0.05）。
结论上调结肠癌HCT-116细胞中miR-7表达能抑制肿瘤细胞的增殖与侵袭，其机制可能与抑制PI3K/AKT信号通路有关。
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关键词:
- miR-7 /
- 结肠癌 /
- 细胞增殖 /
- 凋亡 /
- PI3K/AKT信号通路
Abstract:ObjectiveTo explore the effect of up-regulation of microRNA-7 (miR-7) on biological behavior of colon carcinoma HCT-116 cells and its possible mechanism.
MethodsColon carcinoma HCT-116 cells were divided into miR-7 mimics group (HCT-116 cells transfected with miR-7 mimics), Scramble (negative control) group (HCT-116 cells transfected with miR-Scramble) and MOCD group (HCT-116 cells without transfection). RT-Fq-PCR was used to test the expression of miR-7 at 48h after transfection. CCK-8 assay was used to test the proliferation of HCT-116 cells at 48h after transfection. Cell invasion ability was determined by Transwell assay at 48h after transfection. Cell cycle and apoptosis rate were detected by flow cytometry. The protein levels of PI3K(p110), p-Akt and p-mTOR were examined by Western blot.
ResultsThe expression of miR-7 was significantly up-regulated after transfection with miR-7 mimics(P < 0.05). The proliferation and metastasis of HCT-116 cells were extremely decreased after transfection with miR-7 mimics. FCM showed that the rate of G0/G1 phase was significantly increased, the number of cells in S phase was significantly decreased, the apoptosis rate was significantly upregulated, and the protein levels of PI3K, p-AKT and p-mTOR were decreased in miR-7 mimics group, compared with the other two groups(P < 0.05).
ConclusionUp-regulation of miR-7 could inhibit the proliferation and metastasis of colon carcinoma HCT-116 cells, and the possible mechanism may be related with inhibiting PI3K/AKT signal pathway.
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Key words:
- miR-7 /
- Colon carcinoma /
- Cell proliferation /
- Apoptosis /
- PI3K/AKT signal pathway
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作者贡献马 利： 实验操作，论文撰写 熊兵红 ：实验设计，论文撰写以及修改 黄 卫： 实验操作，论文撰写 罗华友： 论文指导及修改 王昆华： 论文指导及修改
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图 1 上调miR-7对结肠癌HCT-116细胞miR-7表达的影响
Figure 1 Effect of miR-7 mimics on miR-7 expression in HCT-116 cells
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图 2 转染miR-7 mimics后对细胞增殖的影响
Figure 2 Effect of miR-7 mimics on HCT-116 cells proliferation
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图 3 上调miR-7对结肠癌细胞周期的影响
Figure 3 Effect of miR-7 mimics on cell cycle distribution of HCT-116 cells
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图 5 Transwell实验检测上调miR-7对结肠癌细胞侵袭的影响
Figure 5 Effect of miR-7 mimics on HCT-116 cell invasion detected byTranswell assay
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[1] Dai Z, Zheng RS, Zou XN, et al.Analysis and prediction of colorectal cancer incidence trend in China[J]. Zhonghua Yu Fang Yi Xue Za Zhi, 2012, 46(7):598-603. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zhyfyx201207007
[2] Ferlay J, Shin HR, Bray F, et al.Estimates of worldwide burden of cancer in 2008:GLOBOCAN 2008[J]. Int J Cancer, 2010, 127(12):2893-917. http://www.biomedcentral.com/pubmed/21351269
[3] Yang S, Sun Z, Zhou Q, et al.MicroRNAs, long noncoding RNAs, and circular RNAs:potential tumor biomarkers and targets for colorectal cancer[J].Cancer Manag Res, 2018, 10:2249-57. doi: 10.2147/CMAR
[4] To KK, Tong CW, Wu M, et al.MicroRNAs in the prognosis and therapy of colorectal cancer:From bench to bedside[J].World J Gastroenterol, 2018, 24(27):2949-73. doi: 10.3748/wjg.v24.i27.2949
[5] Horsham JL, Ganda C, Kalinowski FC, et al.MicroRNA-7:A miRNA with expanding roles in development and disease[J].Int J Biochem Cell Biol, 2015, 69:215-24. doi: 10.1016/j.biocel.2015.11.001
[6] Webster RJ, Giles KM, Price KJ, et al.Regulation of epidermal growth factor receptor signaling in human cancer cells by microRNA-7[J].J Biol Chem, 2009, 284(9):5731-41. doi: 10.1074/jbc.M804280200
[7] Cao Q, Mao ZD, Shi YJ, et al.MicroRNA-7 inhibits cell proliferation, migration and invasion in human non-small cell lung cancer cells by targeting FAK through ERK/MAPK signaling pathway[J].Oncotarget, 2016, 7(47):77468-81. http://europepmc.org/articles/PMC5363598/
[8] Reddy SD, Ohshiro K, Rayala SK, et al.MicroRNA-7, a homeobox D10 target, inhibits p21-activated kinase 1 and regulates its functions[J].Cancer Res, 2008, 68(20):8195-200. doi: 10.1158/0008-5472.CAN-08-2103
[9] Yue K, Wang X, Wu Y, et al.microRNA-7 regulates cell growth, migration and invasion via direct targeting of PAK1 in thyroid cancer[J].Mol Med Rep, 2016, 14(3):2127-34. doi: 10.3892/mmr.2016.5477
[10] Xu K, Chen Z, Qin C, et al.miR-7 inhibits colorectal cancer cell proliferation and induces apoptosis by targeting XRCC2[J].Oncol Targets Ther, 2014, 7:325-32. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=Doaj000002932472
[11] Liu S, Zhang P, Chen Z, et al.MicroRNA-7 downregulates XIAP expression to suppress cell growth and promote apoptosis in cervical cancer cells[J].FEBS Lett, 2013, 587(14):2247-53. doi: 10.1016/j.febslet.2013.05.054
[12] Xiong S, Zheng Y, Jiang P, et al.MicroRNA-7 inhibits the growth of human non-small cell lung cancer A549 cells through targeting BCL-2[J].Int J Biol Sci, 2011, 7(6):805-14. doi: 10.7150/ijbs.7.805
[13] Wang B, Sun F, Dong N, et al.MicroRNA-7 directly targets insulin-like growth factor 1 receptor to inhibit cellular growth and glucose metabolism in gliomas[J].Diagn Pathol, 2014, 9:211. doi: 10.1186/s13000-014-0211-y
[14] Li Y, Li Y, Liu Y, et al.PAX6, a novel target of microRNA-7, promotes cellular proliferation and invasion in human colorectal cancer cells[J].Dig Dis Sci, 2014, 59(3):598-606. doi: 10.1007/s10620-013-2929-x
[15] Fang Y, Xue JL, Shen Q, et al.MicroRNA-7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3-kinase/Akt pathway in hepatocellular carcinoma[J].Hepatology, 2012, 55(6):1852-62. doi: 10.1002/hep.25576
[16] 徐瑶, 欧阳学农, 陈曦, 等.miR-7抑制喉鳞癌Hep-2细胞增殖及迁移的研究[J].临床肿瘤学杂志, 2015, 12(3):193-7. http://d.old.wanfangdata.com.cn/Periodical/lczlxzz201503001 Xu Y, Ouyang XN, Chen X, et al.Inhibition effect of miR-7 on proliferation and metastasis of Hep-2 laryngeal carcinoma cells[J].Lin Chuang Zhong Liu Xue Za Zhi, 2015, 12(3):193-7. http://d.old.wanfangdata.com.cn/Periodical/lczlxzz201503001
[17] 刘晓智, 苏治国, 姜忠敏, 等.叶酸/聚酰胺-胺介导miR-7治疗小鼠胶质瘤[J].中华肿瘤杂志, 2012, 34(5):325-30. doi: 10.3760/cma.j.issn.0253-3766.2012.05.002 Liu XZ, Su ZG, Jiang ZM, et al.Inhibitory effect of folic acid/polyamide-amine as a miR-7 vector on the growth of glioma in mice[J].Zhonghua Zhong Liu Za Zhi, 2012, 34(5):325-30. doi: 10.3760/cma.j.issn.0253-3766.2012.05.002
[18] 刘亮洪, 张洪云, 刘晓智, 等.脑胶质瘤中miR-7与EGFR/PI3K信号通路相关基因蛋白表达的关系[J].山东医药, 2014, 54(27):1-3. doi: 10.3969/j.issn.1002-266X.2014.27.001 Liu LH, Zhang HY, Liu XZ, et al.Correlation between miR-7 and expression of EGFR/PI3K signal pathway related protein in glioma[J].Shandong Yi Yao, 2014, 54(27):1-3. doi: 10.3969/j.issn.1002-266X.2014.27.001
[19] Yu Z, Ni L, Chen D, et al.Identification of miR-7 as an oncogene in renal cell carcinoma[J].J Mol Histol, 2013, 44(6):669-77. doi: 10.1007/s10735-013-9516-5
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