miR-142-3p靶向FSTL1基因对结直肠癌细胞增殖、凋亡以及放疗敏感性的影响

目的探究miR-142-3p靶向卵泡抑素样蛋白1（FSTL1）对结直肠癌（CRC）细胞增殖、凋亡以及放疗敏感性的影响。 方法培养正常人结肠细胞FHC与CRC细胞系SW480、DLD-1、HCT116和Caco-2，qRT-PCR检测细胞中miR-142-3P水平，Western Blot检测FSTL1蛋白水平；分别转染miR-142-3p模拟物（miR-142-3p组）、模拟物阴性对照（miR-NC组）、FSTL1的si-RNA9（si-FSTL1组）、si-RNA阴性对照（si-con组）至CRCSW480细胞，MTT检测细胞增殖情况，流式细胞仪检测细胞凋亡水平，Western Blot检测增殖、凋亡相关蛋白水平，克隆形成实验检测放射敏感性；双荧光素酶报告基因、Western Blot验证miR-142-3p与FSTL1关系及在CRC中作用。采用t检验和方差分析进行统计学分析。 结果CRC细胞SW480、DLD-?1、HCT116、Caco-2中miR-142-3p水平（0.86±0.09、1.09±0.11、0.76±0.08、0.98±0.10）低于正常结肠细胞FHC （2.56±0.26），差异具有统计学意义（t?= 18.536，15.621，19.851，17.016，P均< 0.01），FSTL1 mRNA水平2.26±0.23、1.39±0.14、2.01±0.20、1.98±0.19高于FHC细胞（0.79±0.08），差异具有统计学意义（t?= 18.110，11.163，16.991，17.317，P均?< 0.01），FSTL1蛋白水平（1.16±0.12、1.09±0.11、0.96±0.09、0.95±0.10）高于FHC细胞（0.37±0.04），差异有统计学意义（t?= 18.736，18.454，17.972，16.155，P均?< 0.01）；miR-?142-?3p组CRC细胞SW480凋亡率（30.23±3.10）和Bax水平（1.02±0.11）高于miR-?con?组8.96±0.89，0.45±0.05，t?= 19.785，14.152，P均< 0.01，72?h细胞增殖活力（1.16±0.11）、CyclinD1 （0.35±0.05）、Bcl-2 （0.38±0.04）、8?Gy （7.56±0.75）细胞存活率低于miR-con组（1.60±0.16，1.02±0.12，0.98±0.10，10.35±1.25，t?= 6.798，15.462，16.713，5.742，P均< 0.01）；si-FSTL1组SW480细胞72?h的细胞增殖活力（1.05±0.11）、CyclinD1（0.40±0.05）、Bcl-2（0.42±0.05）低于si-?con组（1.60±0.16，1.05±0.10，1.00±0.12，t?= 8.498，17.441，13.385，P均< 0.01），Bax（1.00±0.11）高于si-con组（0.41±0.04，t?= 15.122，P?< 0.01）；miR-?142-3p负调控FSTL1表达，过表达FSTL1逆转了miR-142-3p过表达SW480细胞增殖、凋亡及放射敏感性的影响。 结论过表达miR-142-3p可能通过下调FSTL1表达抑制CRC细胞增殖，诱导其凋亡，并增强其放疗敏感性。

Effect of microRNA-142-3p regulation the proliferation,apoptosis and radiosensitivity by targeting FSTL1 in colorectal cancer cells

ObjectiveTo investigate the effects of microRNA-142-3p targeting follistatin-?like protein 1 on proliferation, apoptosis and radiosensitivity of colorectal cancer cells. MethodsThe normal human colon cell FHC and CRC cell lines SW480, DLD-1, HCT116 and Caco-2 were cultured, the level of miR-142-3P was detected by qRT-PCR, and the level of FSTL1 protein was detected by Western Blot. MiR-142-3p mimics (miR-142-3p group) , mimics negative control (miR-NC group) , si-RNA of FSTL1 (si-FSTL1 group) , si-RNA negative control (si-?con group) were transfected into CRC SW480 cells, respectively. MTT was used to detect cell proliferation, flow cytometry was used to detect cell apoptosis, Western Blot was used to detect the levels of proliferation and apoptosis-related proteins, and cloning formation assay was used to detect radiosensitivity. Luciferase and Western Blot were used to verify the relationship between microRNA-142-3p and FSTL1 and its role in colorectal cancer. Statistical analysis was performed using t test and analysis of variance. ResultsThe levels of miR-142-3p in colorectal cancer cells SW480, DLD-1, HCT116, and Caco-2 (0.86±0.09, 1.09±0.11, 0.76±0.08, 0.98±0.10) were lower than those of normal Colonic cells FHC (2.56±0.26, t?= 18.536, 15.621, 19.851, 17.016, P?< 0.01) , FSTL1 mRNA levels (2.26±0.23, 1.39±0.14 , 2.01±0.20, 1.98±0.19) were higher than FHC cell levels (0.79±0.08, t?= 18.110, 11.163, 16.991, 17.317, P?< 0.01) , and FSTL1 protein levels (1.16±0.12, 1.09±0.11, 0.96±0.09, 0.95±0.10) were higher than FHC cell levels (0.37±0.04, t?= 18.736, 18.454, 17.972, 16.155, P?< 0.01) ; the apoptosis rate of colorectal cancer cell SW480 in miR-142-3p group (30.23±3.10) and Bax level (1.02±0.11) were higher than that in miR-con group (8.96±0.89, t?= 19.785, P?< 0.01; 0.45±0.05, t?= 14.152, P?< 0.01) , and cell proliferation activity for 72?h (1.16±0.11) , the levels of CyclinD1 (0.35±0.05) and Bcl-2 (0.38±0.04) , and the cell survival rate after 8?Gy were lower than that of the miR-con group (1.60±0.16, t?= 6.798, P?< 0.01; 1.02±0.12, t?= 15.462, P?< 0.01; 0.98±0.10, t?= 16.713, P?< 0.01; 10.35±1.25, t?= 5.742, P?< 0.01) ; the cell proliferation activity (1.05±0.11) and the levels of CyclinD1 (0.40±0.05) and Bcl-2 (0.42±0.05) in si-FSTL1 group were lower than si-con group (1.60±0.16, t?= 8.498, P?< 0.01; 1.05±0.10, t?= 17.441, P?< 0.01; 1.00±0.12, t?= 13.385, P?< 0.01) , and Bax (1.00±0.11) was higher than si-con group (0.41±0.04, t?= 15.122, P?< 0.01) ; miR-142-3p negatively regulated the expression of FSTL1, and overexpression of FSTL1 reversed the effect of miR-142-3p overexpressing on the proliferation, apoptosis and radiosensitivity of SW480 cells. ConclusionOverexpression of miR-?142-3p may inhibit the proliferation of colorectal cancer cells, induce apoptosis and enhance the sensitivity of radiotherapy by down-regulating FSTL1 expression.