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目的研究微小RNA-543(miR-543)在非小细胞肺癌A549细胞中的表达及其对A549细胞增殖、凋亡和周期的影响和潜在机制。方法用荧光实时定量聚合酶链反应法检测非小细胞肺癌A549细胞与正常肺上皮细胞BEAS-2B中miR-543的表达水平。用miR-543抑制物沉默A549细胞中miR-543表达后,分别将转染miR-543-NC的细胞、miR-543抑制物的细胞、miR-543mimics的细胞命名为正常组、沉默组、过表达组。用细胞增殖试剂盒-8检测miR-543对A549细胞增殖的影响,用流式细胞术检测miR-543对A549细胞凋亡及周期的影响,用荧光素酶报告基因实验检测孕激素与脂联素受体3(PAQR3)是否为miR-543的直接靶分子。结果 miR-543的相对表达量:在人正常肺上皮细胞BEAS-2B和肺癌细胞系A549中分别为1.00±0.05,3.87±0.04,两者比较差异有统计学意义(P<0.05)。正常组与沉默组的A549细胞中miR-543的相对表达量分别为1.00±0.05,0.42±0.07,差异有统计学意义(P<0.05),说明miR-543 inhibitors可抑制A549细胞中的miR-543表达。连续5 d OD值:正常组和沉默组的细胞分别为0.25±0.05,0.48±0.04,0.73±0.05,0.99±0.05,1.35±0.04;0.25±0.04,0.36±0.04,0.47±0.05,0.58±0.05,0.66±0.04,组间比较差异均有统计学意义(均P<0.05),说明miR-543抑制物可显著抑制A549细胞增殖。A549细胞凋亡率和A549细胞早期凋亡率:正常组和沉默组分别为(1.21±0.37)%,(5.78±1.69)%;(0.84±0.22)%,(2.18±1.28)%,组间比较差异均有统计学意义(均P<0.05),说明miR-543抑制物可显著促进A549细胞凋亡和早期凋亡。A549细胞G1期细胞百分比:正常组和沉默组分别为(51.27±3.19)%,(59.30±4.19)%,组间比较差异有统计学意义(P<0.05),说明miR-543抑制物阻滞A549细胞周期于G1期。过表达组细胞与野生型(WT)孕激素PAQR3和与突变型PAQR3(mutant)相结合的荧光素酶报告基因的相对活性分别为0.42±0.05,0.97±0.04,两者比较差异有统计学意义(P<0.05);而正常组分别为1.00±0.05,0.94±0.07,两者比较差异无统计学意义,说明PAQR3为miR-543的直接靶分子,miR-543可在A549细胞中直接调控PAQR3的表达。结论 miR-543通过直接靶向抑制PAQR3的表达而调控非小细胞肺癌A549的增殖、凋亡与周期。
Objective To study the expression of miR-543 (miR-543) in non-small cell lung cancer A549 cells and its effect on A549 cell proliferation, apoptosis and cell cycle. Methods The expression of miR-543 in non-small cell lung cancer A549 cells and normal lung epithelial cells BEAS-2B was detected by real-time fluorescence quantitative polymerase chain reaction. After miR-543 expression was silenced in A549 cells with miR-543 inhibitor, the cells transfected with miR-543-NC, miR-543 inhibitor and miR-543mimics were named as normal group and silencing group Expression group. The effect of miR-543 on the proliferation of A549 cells was detected by using cell proliferation kit-8. Flow cytometry was used to detect the effect of miR-543 on the apoptosis and the cycle of A549 cells. Luciferase reporter assay was used to detect the relationship between progesterone and adiponectin Receptor 3 (PAQR3) is a direct target of miR-543. Results The relative expression level of miR-543 was 1.00 ± 0.05,3.87 ± 0.04 in human normal epithelial cells BEAS-2B and lung cancer cell line A549, respectively. The difference was statistically significant (P <0.05). The relative expression levels of miR-543 in A549 cells in normal and silent groups were 1.00 ± 0.05 and 0.42 ± 0.07, respectively (P <0.05), indicating that miR-543 inhibitors could inhibit the expression of miR- 543 expression. For 5 days, the OD values were 0.25 ± 0.05, 0.48 ± 0.04, 0.73 ± 0.05, 0.99 ± 0.05, 1.35 ± 0.04, 0.25 ± 0.04, 0.36 ± 0.04, 0.47 ± 0.05 and 0.58 ± 0.05 , 0.66 ± 0.04, the difference between the two groups was statistically significant (all P <0.05), indicating that miR-543 inhibitor can significantly inhibit A549 cell proliferation. The apoptosis rate of A549 cells and early apoptosis of A549 cells were (1.21 ± 0.37)%, (5.78 ± 1.69)%, (0.84 ± 0.22)% and (2.18 ± 1.28)% respectively in the normal group and the silent group (All P <0.05), indicating that miR-543 inhibitor can significantly promote A549 cell apoptosis and early apoptosis. The percentages of G1 phase cells in A549 cells were (51.27 ± 3.19)% and (59.30 ± 4.19)%, respectively, with significant difference between the two groups (P <0.05), indicating that miR-543 inhibitor block A549 cell cycle in G1 phase. The relative activity of luciferase reporter gene combined with wild-type (WT) progestin PAQR3 and mutant PAQR3 (mutant) was 0.42 ± 0.05 and 0.97 ± 0.04, respectively, with significant difference between the two groups (P <0.05), while the normal control group was 1.00 ± 0.05 and 0.94 ± 0.07, respectively. There was no significant difference between the two groups (P> 0.05), indicating that PAQR3 was a direct target of miR-543 and miR-543 could directly regulate PAQR3 expression. Conclusion miR-543 regulates the proliferation, apoptosis and cycle of non-small cell lung cancer A549 cells by directly targeting PAQR3 expression.