摘要目的 探讨微RNA(miR)?16?5p调控尾侧同源盒基因(CDX2)蛋白表达对白血病细胞增殖及凋亡的影响及其作用机制.方法 实时荧光定量聚合酶链反应(qRT?PCR)与蛋白免疫印迹分别检测不同白血病细胞及正常人外周血单核细胞(PBMC)中miR?16?5p与CDX2表达水平;双荧光素酶实验检测miR?16?5p与CDX2之间的相互关系;MTT实验检测miR?16?5p过表达或抑制CDX2表达对K562细胞增殖的影响;流式细胞术检测miR?16?5p过表达或抑制CDX2表达对K562细胞凋亡的影响;蛋白免疫印迹检测CyclinD1、Bcl?2、Bax、p21蛋白表达;MTT实验与流式细胞术检测过表达CDX2后对K562细胞增殖及凋亡的逆转作用.结果 与PBMC细胞相比,不同白血病细胞中miR?16?5p表达明显降低,CDX2表达明显升高(均P<0.05);与miR?NC组比较,miR?16?5p组野生型WT?CDX2荧光素酶活性明显降低(0.49±0.05比1.05±0.08,P<0.001),与突变型MUT?CDX2荧光素酶活性略有升高(1.06±0.09比1.04±0.07,P>0.05),miR?16?5p可调控CDX2蛋白表达水平;MTT实验和流式细胞术实验显示培养48、72 h时miR?16?5p组、si?CDX2组K562细胞A值明显低于miR?NC组、si?NC组,均P<0.05),K562细胞的凋亡率明显升高[(21.54 ± 2.17)%比(6.83 ± 0.67)% ,(19.38 ± 1.85)%比(7.24 ± 0.71)% ,均P<0.05)], CyclinD1(0.32±0.04比0.71±0.07,0.38±0.04比0.75±0.07)与Bcl?2蛋白(0.33±0.03比0.76±0.08,0.36± 0.04比0.79±0.07)表达明显下调(均P<0.05),Bax(0.78±0.08比0.32±0.03,0.75±0.07比0.26±0.03)与p21蛋白(0.79±0.06比0.28±0.03,0.77±0.07比0.24±0.03)表达明显上调(均P<0.05);CDX2过表达可逆转miR?16?5p对白血病细胞增殖的抑制作用及其对细胞凋亡的促进作用.结论 miR?16?5p可靶向调控CDX2蛋白抑制白血病细胞增殖并诱导细胞凋亡.

abstractsObjective To investigate the effect of caudal?type homeobox transcription factor 2 (CDX2) protein expression regulated by microRNA(miR)?16?5p on the proliferation and apoptosis of leukemia cells and its mechanism. Methods Real?time quantitative polymerase chain reaction(qRT?PCR) and Western blot were used to determine the expression levels of miR?16?5p and CDX2 in different leukemia cells and normal peripheral blood mononuclear cells(PBMC),respectively. Dual?luciferase assay was used to determine the relationship between miR?16?5p and CDX2. MTT assay was used to determine the effect of miR?16?5p overexpression or inhibition of CDX2 expression on the proliferation of K562 cells. Flow cytometry was used to determine the effect of miR?16?5p overexpression or inhibition of CDX2 expression on the apoptosis of K562 cells. Western blot was used to determine the protein expression of CyclinD1,Bcl?2, Bax and p21. MTT assay and flow cytometry were used to determine the reversal of proliferation and apoptosis of K562 cells after overexpression of CDX2. Results Compared with PBMC cells,the expression level of miR?16?5p in different leukemia cells significantly decreased,whereas the expression level of CDX2 significantly increased (both P<0.05). Dual?luciferase reporter assay system showed that miR?16?5p regulated the protein expression level of CDX2. MTT assay and flow cytometry showed that the A value of K562 cells in miR?16?5p group and si?CDX2 group was significantly lower than that in miR?NC group and si?NC group at 48 and 72 h(all P<0.05). The apoptosis rate of K562 cells significantly increased[(21.54± 2.17)% vs(6.83 ± 0.67)% ,(19.38 ± 1.85)% vs(7.24 ± 0.71)% ,both P<0.05]. The protein expression levels of CyclinD1(0.32±0.04 vs 0.71±0.07,0.38±0.04 vs 0.75±0.07)and Bcl?2(0.33±0.03 vs 0.76± 0.08,0.36± 0.04 vs 0.79 ± 0.07)were significantly down?regulated(all P<0.05). The protein expression levels of Bax(0.78±0.08 vs 0.32±0.03,0.75±0.07 vs 0.26±0.03)and p21(0.79±0.06 vs 0.28±0.03,0.77± 0.07 vs 0.24 ± 0.03)were significantly up?regulated(all P<0.05). Overexpression of CDX2 reversed the inhibiton and promotion of miR?16?5p on proliferation and apoptosis of leukemia cells,respectively. Conclusion miR?16?5p may regulate CDX2 protein to inhibit leukemia cell proliferation and induce apoptosis.