目的探讨微小RNA-3917(miR-3917)对肺癌细胞增殖及对生物钟基因Period2(PER2)表达的影响.方法取复苏后的肺腺癌A549细胞培养至对数生长期,采用脂质体法向A549细胞转染10、20、50 nmol/L靶向miR-3917的 siRNA表达载体pEGFP-miR-3917-siRNA质粒,转染48 h后荧光显微镜下观察绿色荧光蛋白的表达;根据实验设计分为对照组、空载体组和干扰组,空载体组及干扰组的质粒浓度均为50 nmol/L,采用实时荧光定量PCR(QPCR)检测转染48 h后的 miR-3917相对表达量,QPCR和Western blotting分别检测转染48 h后的PER2 mRNA和蛋白水平,采用CCK-8法检测各组转染24、48、72 h的A549细胞增殖抑制率,双荧光素酶靶标实验验证miR-3917与PER2的相互关系.结果QPCR检测发现对照组、空载体组和干扰组的miR-3917相对表达量依次为1.007±0.018、1.068±0.084和0.171±0.052,干扰组的miR-3917相对表达量低于对照组和空载体组,差异有统计学意义(P<0.05).对照组、空载体组和干扰组的PER2 mRNA相对表达量依次为1.060±0.129、1.086±0.229和2.920±0.927,蛋白表达量依次为0.204±0.046、0.183±0.043和0.512±0.117,干扰组的 PER2 mRNA和蛋白水平均高于对照组和空载体组,差异有统计学意义(P<0.05).与对照组和空载体组比较,干扰组的A549细胞增殖能力下降,转染24、48、72h后的增殖抑制率依次为(34.192±5.268)％、(33.527±6.603)％和(30.591±7.788)％,均高于其余两组,差异有统计学意义(P<0.05).miR-3917可显著抑制野生型PER2-3' UTR质粒转染细胞的荧光素酶活性,而对突变型PER2-3'UTR质粒转染细胞的荧光素酶活性并无影响.结论下调miR-3917可抑制肺癌细胞增殖并升高PER2水平, miR-3917对PER2有靶向调控作用,可作为肺癌防治的新靶点.%Objective To investigate the effect of microRNA-3917 (miR-3917) on the proliferation of lung cancer cells and the expression of biological clock gene Period2 (PER2). Methods The recovered A549 cells were cultured to logarithmic growth phase. The pEGFP-miR-3917-siRNA plasmid vector targeting miR-3917 was transfected into A549 cells by 10, 20 and 50 nmol/L liposome. The expression of green fluorescent protein was observed under fluorescence microscope after 48 h transfection. According to the experimental design, they were divided into control group, empty load group and interference group. The plasmid concentration in empty load group and interference group was 50 nmol/L. The real-time quantitative PCR (QPCR) was used to detect the relative expression of miR-3917 at 48 h after transfection. The mRNA and protein levels of PER2 were detected by QPCR and Western blotting after at 48 h after transfection, respectively. The inhibitive rates of proliferation at 24, 48 and 72 h after transfection were detected by CCK-8 assay. Dual luciferase target assay was employed to verify that PER2 was a direct target of miR-3917. Results The relative expression levels of miR-3917 in control group, empty load group and interference group were 1.007±0.018,1.068±0.084 and 0.171±0.052. The relative expression level of miR-3917 in the interference group was lower than that in the control group and empty load group, and the difference was statistically significant (P<0.05). The relative expression levels of PER2 were 1.060±0.129, 1.086±0.229 and2.920±0.927 in the control group, empty load group and interference group, and the protein levels were 0.204±0.046, 0.183±0.043 and 0.512±0.117. The mRNA and protein levels of PER2 in the interference group were higher than those in the control group and empty load group, and the difference was statistically significant (P<0.05). Compared with the control group and empty load group, the proliferative ability decreased in interference group. The inhibitive rates of proliferation were (34.192±5.268)％, (33.527±6.603)％ and (30.591±7. 788)％ at 24,48,72 h in interference group, higher than other two groups, and the difference was statistically significant (P<0.05). miR-3917 could significantly inhibit the luciferase activity of wild-type PER2-3' UTR plasmid transfected cells, but had no significant effect on the luciferase activity of mutant PER2-3 ' UTR plasmid transfected cells. Conclusion Downregulation of miR-3917 can inhibit the proliferation of lung cancer cells and increase the expression of PER2. miR-3917 has a targeted regulation effect on PER2, and can be used as a new target for the prevention and treatment of lung cancer.
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