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目的通过小干扰RNA(siRNA)沉默K562细胞bcr/abl基因使SH2肌醇磷脂酶(SHIP)基因表达增加,探讨SHIP基因缺失对K562细胞增殖凋亡的影响及机制。方法构建bcr/abl基因的化学合成siRNA,并转染到K562细胞,分为转染特异性siRNA实验组、转染非特异性siRNA对照组、空白对照组。Western blotting法检测转染后K562细胞p210表达量,实时荧光定量PCR(FQ-RT-PCR)检测各组细胞中SHIP mRNA表达量;Western blotting法检测SHIP、磷酸化蛋白激酶B 308(p-Akt308)和磷酸化蛋白激酶B 473(p-Akt473)的表达量;MTT比色法检测K562细胞增殖率;流式细胞仪检测K562细胞凋亡率;酶联免疫吸附试验(ELISA)法检测核因子(NF)-κB活性。结果转染特异性siRNA实验组K562细胞p210表达量、p-Akt308表达量、p-Akt473表达量较空白对照组和转染非特异性siRNA对照组降低,SHIP mRNA表达量、SHIP表达量、细胞凋亡率较空白对照组和转染非特异性siRNA对照组升高(P<0.05);空白对照组与转染非特异性siRNA对照组K562细胞p210表达量、SHIP mRNA表达量、SHIP表达量、p-Akt308表达量、p-Akt473表达量、细胞凋亡率比较,差异无统计学意义(P>0.05)。第1天、第3天、第5天转染特异性siRNA实验组K562细胞增殖率较空白对照组和转染非特异性siRNA对照组降低(P<0.05);空白对照组与转染非特异性siRNA对照组K562细胞增殖率比较,差异无统计学意义(P>0.05)。第3天、第5天转染特异性siRNA实验组NF-κB活性较空白对照组和转染非特异性siRNA对照组降低(P<0.05);空白对照组与转染非特异性siRNA对照组NF-κB活性比较,差异无统计学意义(P>0.05)。结论 SHIP基因表达受bcr/abl基因的调控,SHIP表达缺失可能是K562细胞过度增殖和凋亡减少的重要因素。
Objective To investigate the effects of SHIP gene deletion on the proliferation and apoptosis of K562 cells by silencing the bcr / abl gene of K562 cells with small interfering RNA (siRNA) to increase SH2 inositol phospholipase (SHIP) gene expression. Methods The chemically synthesized siRNA of bcr / abl gene was constructed and transfected into K562 cells. The transfected siRNA was transfected into nonspecific siRNA control group and blank control group. The expression of p21 in K562 cells was detected by Western blotting and the expression of SHIP mRNA in each group was detected by real-time fluorescence quantitative PCR (FQ-RT-PCR). The expressions of SHIP, p-Akt308 ) And phosphorylated protein kinase B 473 (p-Akt473). The proliferation rate of K562 cells was detected by MTT colorimetric assay. The apoptosis rate of K562 cells was detected by flow cytometry. The expressions of nuclear factor (NF) -κB activity. Results The expression of p210, the expression of p-Akt308 and the expression of p-Akt473 in K562 cells transfected with specific siRNA were lower than those in the blank control and non-specific siRNA transfected groups. The expression of SHIP mRNA, (P <0.05). Compared with blank control group and non-specific siRNA transfected group, the expression of p210, SHIP mRNA, SHIP mRNA and protein in K562 cells were significantly increased Akt308 expression, p-Akt473 expression, apoptosis rate comparison, the difference was not statistically significant (P> 0.05). The proliferation rate of K562 cells transfected with specific siRNA on the 1st day, the 3rd day and the 5th day was lower than that of the blank control group and the non-specific siRNA transfected group (P <0.05). The blank control group and the transfected non-specific siRNA The proliferation rate of K562 cells in control group showed no significant difference (P> 0.05). The activity of NF-κB in transfected specific siRNA group was lower than that in blank control group and non-specific siRNA transfected group on day 3 and day 5 (P <0.05). Compared with blank control group and untransfected siRNA group, NF- κB activity, the difference was not statistically significant (P> 0.05). Conclusion The expression of SHIP gene is regulated by bcr / abl gene. The absence of SHIP expression may be an important factor in the over-proliferation and apoptosis of K562 cells.