CELF1通过抑制CDKN1B促进胶质瘤细胞增殖的研究

CELF1 promotes proliferation of glioma by inhibiting CDKN1B

目的探讨CELF1在胶质瘤细胞增殖中的作用及可能机制。方法采用实时荧光定量PCR(QPCR)和Western blotting检测胶质瘤细胞系U87、U251、SHG44以及人正常星形胶质细胞系HA1800中CELF1 m RNA和蛋白表达;无义核酸NC(NC组)、si CELF1(si CELF1组)和si CELF1&siCDKN1B(si CELF1&siCDKN1B组)转染U87细胞,CCK-8法检测细胞活力,流式细胞术检测细胞周期变化; Western blotting检测CELF1、CDKN1B和Cyclin D1蛋白表达。结果胶质瘤细胞系U87、U251和SHG44中CELF1 m RNA相对表达量分别为3. 1±0. 074、4. 2±0. 137和3. 6±0. 023,均高于人正常星形胶质细胞HA1800的1. 1±0. 054,差异均有统计学意义(P <0. 05)。Western blotting检测CELF1的蛋白表达与m RNA表达一致。沉默CELF1能显著降低U87细胞活力,si CELF1组G0/G1期细胞比例为(63. 5±1. 33)%,显著高于NC组的(39. 4±1. 24)%,而si CELF1组S期细胞比例为(14. 3±0. 095)%,显著低于NC组的(22. 8±1. 97)%(P<0. 05)。si CELF1组CDKN1B蛋白相对表达量为2. 37±0. 088,显著低于NC组的1. 17±0. 101(P<0. 05)。si CELF1组Cyclin D1蛋白相对表达量为0. 274±0. 039,显著低于si CELF1&siCDKN1B组的0. 83±0. 071 (P <0. 05)。si CELF1&siCDKN1B组细胞活力高于si CELF1组(P <0. 05)。si CELF1&siCDKN1B组G0/G1期细胞比例为(42. 1±1. 76)%,显著低于si CELF1组的(62. 4±1. 92)%(P <0. 05);而si CELF1&siCDKN1B组S期细胞比例为(20. 3±0. 077)%,与NC组的(22. 8±1. 97)%差异无统计学意义。结论 CELF1通过抑制CDKN1B,进而介导Cyclin D1表达促进胶质瘤细胞增殖。

Objective To investigate the role and possible mechanism of CELF1 in the proliferation of glioma cells.Methods CELF1 mRNA and protein expression in glioma cell lines U87, U251, SHG44 and human normal astrocyte line HA1800 was detected by fluorescence quantitative PCR （QPCR） and Western blotting. U87 cells were transfected with nonsense nucleic acid NC （NC group）, siCELF1 （siCELF1 group） and siCELF1＆siCDKN1B （siCELF1＆siCDKN1B group）. Cell viability was detected by CCK-8 assay, and cell cycle changes were detected by flow cytometry. The protein expression of CELF1, CDKN1B and Cyclin D1 was detected by Western blotting.Results The relative expression levels of CELF1 mRNA in glioma cell lines U87, U25 1 and SHG44 were 3.1±0.074, 4.2±0.137 and 3.6±0.023, respectively, which were higher than those in normal astrocyte line HA 1800, and the differences were statistically significant （ P 〈0.05）. The protein expression of CELF1 detected by Western blotting was consistent with that of mRNA. Silence CELF1 can significantly reduce U87 cell viability. The proportion of G 0/G 1 phase cells in siCELF1 group was （63.5±1.33）%, significantly higher than that in NC group （39.4±1.24）%;while proportion of S phase cells in siCELF1 group was （14.3±0.095）%, significantly lower than that in NC group （22.8±1.97）% （ P 〈0.05）. The relative expression of CDKN1B protein in group siCELF1 was 2.37±0.088, which was significantly lower than that in NC group （ P 〈0.05）. The relative expression of Cyclin D1 protein in group siCELF1 was 0.274±0.039, which was significantly lower than that in siCELF1＆siCDKN1B group （ P 〈0.05）. Cell viability in group siCELF1＆siCDKN1B was higher than that in group siCELF1 （ P 〈0.05）. The proportion of G 0/G 1 phase cells in siCELF1-siCDKN1B group was （42.1±1.76）%, significantly lower than that in siCELF1 group （62.4±1.92）% （ P 〈0.05）, while the proportion of S phase cells in siCELF1-siCDKN1B group was （20.3±0.077）%, which was not significantly different from that in NC group （22.8±1.97）%.Conclusion CELF1 mediates the expression of Cyclin D1 by inhibiting CDKN1B, and promotes glioma proliferation.