miR-215-3p通过调控CXCR4逆转结肠癌HCT8细胞5-FU耐药的实验研究
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摘要
目的 探讨miR-215-3p对CXC结构趋化因子受体4(CXCR4)及CXCR4/RhoA信号通路的调控作用,以及对结肠癌HCT8细胞5-FU耐药的影响。方法 体外培养HCT8细胞和HCT8/5-FU耐药细胞分别作为HCT8组和HCT8/5-FU组,将miR-215-3p mimics转染至HCT8/5-FU细胞作为miR-215-3p组,转染无义序列为NC组。采用MTT法检测各组细胞增殖能力的改变,计算半数抑制浓度(IC_(50))。采用双荧光素酶报告实验验证miR-215-3p与CXCR4的靶向关系。采用Western blotting法检测各组CXCR4/RhoA信号通路相关蛋白CXCR4、RhoA、PI3K、RhoGEF、PAK、PKN1和ROCK的表达水平。结果 5-FU对HCT8细胞和HCT8/5-FU细胞的IC_(50)分别为19.98μmol/L和105.86μmol/L。HCT8/5-FU的耐药指数为5.30。HCT8/5-FU组miR-215-3p表达水平为0.54±0.13,低于HCT8组(P<0.05)。miR-215-3p组miR-215-3p表达水平为1.49±0.22,高于NC组和HCT8/5-FU组(P<0.05)。1、3、9、27、81μmol/L 5-FU对miR-215-3p组HCT8/5-FU细胞增殖抑制率分别为(14.46±1.79)%、(20.37±2.64)%、(54.87±3.81)%、(79.51±3.67)%和(84.62±4.15)%;相同浓度下, miR-215-3p组细胞增殖抑制率高于HCT8/5-FU组和NC组(P<0.05)。miR-215-3p可抑制野生型CXCR4 3’-UTR报告基因载体的荧光素酶活性,而对突变型CXCR4 3’-UTR的荧光素酶活性无影响。miR-215-5p组CXCR4、RhoA、PI3K、RhoGEF、PAK、PKN1和ROCK蛋白表达水平分别为0.41±0.07、0.34±0.08、0.82±0.15、0.29±0.04、0.39±0.06、0.30±0.03和0.51±0.08,均低于其余3组,差异有统计学意义(P<0.05)。结论 上调miR-215-3p表达可逆转HCT8/5-FU细胞株对5-FU的耐药性,其可能的作用机制可能与负性调控CXCR4/RhoA通路的活性有关。
Objective To investigate the regulatory effect of miR-215-3 p on CXC structural chemokine receptor 4(CXCR4) and CXCR4/RhoA signaling pathway and on 5-FU resistance in colon cancer cells. Methods HCT8 cells and HCT8/5-FU resistant cells were cultured in vitro as HCT8 group and HCT8/5-FU group, respectively. miR-215-3 p mimics was transfected into HCT8/5-FU cells as miR-215-3 p group, and the unintentional sequence was transfected as NC group. MTT assay was used to detect the change of cell proliferation ability in each group, and the half inhibitory concentration(IC_(50)) was calculated. Double luciferase reporter assay was used to verify the targeting relationship between miR-215-3 p and CXCR4. The expression levels of CXCR4/RhoA signaling pathway related proteins CXCR4, RhoA, PI3 K, RhoGEF, PAK, PKN1 and ROCK were detected by Western blotting method. Results The IC_(50) of 5-FU on HCT8 cells and HCT8/5-FU cells were 19.98 μmol/L and 105.86 μmol/L, respectively. The resistance index of HCT8/5-FU was 5.30. The expression level of miR-215-3 p in HCT8/5-FU group was 0.54±0.13, lower than that in HCT8 group(P<0.05). The expression level of miR-215-3 p in miR-215-3 p group was 1.49±0.22, higher than that in group of NC and HCT8/5-FU(P<0.05). The inhibition rates of 1, 3, 9, 27 and 81 μmol/L 5-FU on the proliferation of HCT8/5-FU cells in the miR-215-3 p group were(14.46±1.79)%,(20.37±2.64)%,(54.87±3.81)%,(79.51±3.67)% and(84.62 ±4.15)% respectively. At the same concentration, the inhibition rate of cell proliferation in the group of miR-215-3 p was higher than that in the group of HCT8/5-FU and NC(P<0.05). miR-215-3 p inhibited the luciferase activity of wild type CXCR4 3'-UTR reporter gene vector, but had no effect on mutant CXCR4 3'-UTR luciferase activity. The expressions of CXCR4, RhoA, PI3 K, RhoGEF, PAK, PKN1 and ROCK proteins in miR-215-3 p group were respectively 0.41±0.07, 0.34±0.08, 0.82±0.15, 0.29±0.04, 0.39±0.06, 0.30±0.03 and 0.51±0.08, which were lower than these proteins levels than HCT8/5-FU group, HCT8 group and NC group(P<0.05). Conclusion Upregulation of the expression of miR-215-3 p can reverse the resistance of HCT8/5-FU cell lines to 5-FU, and its possible mechanism may be related to the negative regulation of the activity of CXCR4/RhoA pathway.
Objective To investigate the regulatory effect of miR-215-3 p on CXC structural chemokine receptor 4(CXCR4) and CXCR4/RhoA signaling pathway and on 5-FU resistance in colon cancer cells. Methods HCT8 cells and HCT8/5-FU resistant cells were cultured in vitro as HCT8 group and HCT8/5-FU group, respectively. miR-215-3 p mimics was transfected into HCT8/5-FU cells as miR-215-3 p group, and the unintentional sequence was transfected as NC group. MTT assay was used to detect the change of cell proliferation ability in each group, and the half inhibitory concentration(IC_(50)) was calculated. Double luciferase reporter assay was used to verify the targeting relationship between miR-215-3 p and CXCR4. The expression levels of CXCR4/RhoA signaling pathway related proteins CXCR4, RhoA, PI3 K, RhoGEF, PAK, PKN1 and ROCK were detected by Western blotting method. Results The IC_(50) of 5-FU on HCT8 cells and HCT8/5-FU cells were 19.98 μmol/L and 105.86 μmol/L, respectively. The resistance index of HCT8/5-FU was 5.30. The expression level of miR-215-3 p in HCT8/5-FU group was 0.54±0.13, lower than that in HCT8 group(P<0.05). The expression level of miR-215-3 p in miR-215-3 p group was 1.49±0.22, higher than that in group of NC and HCT8/5-FU(P<0.05). The inhibition rates of 1, 3, 9, 27 and 81 μmol/L 5-FU on the proliferation of HCT8/5-FU cells in the miR-215-3 p group were(14.46±1.79)%,(20.37±2.64)%,(54.87±3.81)%,(79.51±3.67)% and(84.62 ±4.15)% respectively. At the same concentration, the inhibition rate of cell proliferation in the group of miR-215-3 p was higher than that in the group of HCT8/5-FU and NC(P<0.05). miR-215-3 p inhibited the luciferase activity of wild type CXCR4 3'-UTR reporter gene vector, but had no effect on mutant CXCR4 3'-UTR luciferase activity. The expressions of CXCR4, RhoA, PI3 K, RhoGEF, PAK, PKN1 and ROCK proteins in miR-215-3 p group were respectively 0.41±0.07, 0.34±0.08, 0.82±0.15, 0.29±0.04, 0.39±0.06, 0.30±0.03 and 0.51±0.08, which were lower than these proteins levels than HCT8/5-FU group, HCT8 group and NC group(P<0.05). Conclusion Upregulation of the expression of miR-215-3 p can reverse the resistance of HCT8/5-FU cell lines to 5-FU, and its possible mechanism may be related to the negative regulation of the activity of CXCR4/RhoA pathway.
引文
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[13] Sheng X,Zhong H,Wan H,et al.Granulocyte colony-stimulating factor inhibits CXCR4/SDF-1α signaling and overcomes stromal-mediated drug resistance in the HL-60 cell line[J].Exp Ther Med,2016,12(1):396-404.
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[15] Cabrero-de Las Heras S,Martínez-Balibrea E.CXC family of chemokines as prognostic or predictive biomarkers and possible drug targets in colorectal cancer[J].World J Gastroenterol,2018,24(42):4738-4749.
[2] Wu N,Xu L,Yang Y,et al.Tetramethylpyrazine-mediated regulation of CXCR4 in retinoblastoma is sensitive to cell density[J].Mol Med Rep,2017,15(5):2481-2488.
[3] Burger JA,Kipps TJ.CXCR4:a key receptor in the crosstalk between tumor cells and their microenvironment [J].Blood,2006 ,107(5):1761-1767.
[4] Lan WW,Chen SL,Tong L.MicroRNA-215 regulates fibroblast function:insights from a human fibrotic disease[J].Cell Cycle,2015,14(12):1973-1984.
[5] Karaayvaz M,Pal K,Song B,et al.Prognostic Significance of miR-215 in colon cancer[J].Clin Colorectal Cancer,2011,10(4):340-347.
[6] 黄鹏,王国琴,董泽涛,等.SDF-1α/CXCR7介导结肠癌细胞对5-FU的耐药作用[J].肿瘤,2018,38(1):18-24.
[7] 苏建伟,周喜汉.结肠癌细胞耐药的研究现状[J].右江医学,2017,42(1):91-4.
[8] Agostini A,Brunetti M,Davidson B,et al.The microRNA miR-192/215 family is upregulated in mucinous ovarian carcinomas[J].Sci Rep,2018,8(1):11069.
[9] Chen Z,Han S,Huang W,et al.MicroRNA-215 suppresses cell proliferation,migration and invasion of colon cancer by repressing Yin-Yang 1[J].Biochem Biophys Res Commun,2016,479(3):482-488.
[10] Li XW,Qiu SJ,Zhang X,et al.Overexpression of miR-215-3p sensitizes colorectal cancer to 5-fluorouracil induced apoptosis through regulating CXCR1[J].Eur Rev Med Pharmacol Sci,2018,22(21):7240-7250.
[11] 尹凌帝,孙倩,孙明,等.人类癌症中长链非编码RNA与miRNA相互作用的研究进展[J].临床肿瘤学杂志,2014,19(7):662-666.
[12] 陈力,金玉麟,王琳,等.miRNA与肺癌顺铂耐药相关性的研究进展[J].复旦学报(医学版),2018,48(2):245-249.
[13] Sheng X,Zhong H,Wan H,et al.Granulocyte colony-stimulating factor inhibits CXCR4/SDF-1α signaling and overcomes stromal-mediated drug resistance in the HL-60 cell line[J].Exp Ther Med,2016,12(1):396-404.
[14] Yin H,Wang Y,Chen W,et al.Drug-resistant CXCR4-positive cells have the molecular characteristics of EMT in NSCLC[J].Gene,2016,594(1):23-29.
[15] Cabrero-de Las Heras S,Martínez-Balibrea E.CXC family of chemokines as prognostic or predictive biomarkers and possible drug targets in colorectal cancer[J].World J Gastroenterol,2018,24(42):4738-4749.
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