肌醇5’磷酸酶基因突变对K562细胞周期蛋白和Akt磷酸化的影响

Effects of SHIP gene mutation on cell cycle related proteins and phosphorylated Akt in K562 cells

目的从分子水平探讨肌醇5’磷酸酶（SHIP）基因突变对人白血病细胞系K562细胞周期及其相关基因表达的影响。方法应用携带野生型和突变型SHIP及绿色荧光蛋白的慢病毒及空载体慢病毒质粒转染K562细胞，通过流式细胞术检测K562／SHIP细胞转染效率、细胞增殖指数及细胞周期变化；MTT法检测细胞增殖活性改变，实时荧光定量PCR（FQ—PCR）检测SHIPmRNA水平变化，Western blot检测各组K562细胞SHIP、细胞周期蛋白（cyclin）D1、p21^WAF1/CIPI、p27^KIP1蛋白表达水平及Akt磷酸化变化。结果野生型SHIP基因能明显抑制K562细胞增殖，并产生明显的G0／G1期阻滞[G0／G1期细胞分别为（34．2±7．8）％和（0．7±8．3）％，P〈0．01]；但SHIP基因点突变并未影响K562细胞增殖及细胞周期改变[G0／G1期细胞分别为（33．4±4．2）％和（36．3±6．7）％，P〉0．05]。Westernblot结果发现转染野生型SHIP基因后K562细胞Akt磷酸化和cyclin D1表达水平明显下降（P〈0．01），p21^WAF1/CIPI、p27^KIP1表达增高，而突变型SHIP基因对Akt磷酸化水平和细胞周期蛋白表达几乎没有影响（P〉0．05）。结论①野生型SHIP基因通过下调K562细胞Akt磷酸化水平，进而抑制其下游cyclinD1表达，上调p21^WAF1/CIPI和p27^KIP1基因表达，导致细胞周期阻滞在G0／G1期，抑制K562细胞增殖；②SHIP基因突变体（TTC→CTC，Phe→Leu）丧失了对K562细胞的增殖抑制作用，提示SHIP基因对K562细胞增殖的负调控作用有赖于其基因结构和功能正常。

Objective To investigate the effect of SHIP gene mutation on the cell cycle and its related gene expression in K562 cells. Methods The recombinated green fluorescent protein （GFP） containing FIV-SHIP gene was transfected into K562 cells. The transfection efficiency and cell cycle of K562/SHIP were assessed by flow cytometry （FCM）. The proliferation of K562 cells was detected by MTT assay, the mRNA levels of SHIP by real-time fluorescent relative-quantification reverse transcriptional PCR（ FQ- PCR）, and the protein levels of SHIP, CycliuD1,p21^WAF1/CIPIandp27^KIP1 by Western blot. Results Wild type SHIP inhibited K562 cell proliferation and caused a G0/G1 arrest [ （ 34.2 ± 7.8 ） % vs （0.7 ± 8.3 ） % （ P 〈 0.01 ） ] ; while the point mutation of SHIP gene did not show such effect. Western blot results showed that the Akt phosphorylation and cyclin DI expression was significantly decreased （ P 〈 0.01 ） , and the expression ofp21^WAF1/CIPIandp27^KIP1increased. Site-directed mutation of SHIP gene SH2 domain （TTC→CTC, Phe→Leu） did not influence the Akt phosphorylation and cyclins （ P〉0.05 ）. Conclusion （1）wtSHIP gene can downregulate Akt phosphorylation and result in inhibition of cyclin D1 expression, up-regulating p27KIPI andp21^WAF1/CIPI expression, finally leading to the reduction of K562 cell proliferation, and inducing G0/G1 phase arrest. （2）SHIP gene suppresses the proliferation of K562, being dependent on its intact structure and function.