CRKⅡ对慢性粒细胞白血病K562细胞恶性行为的影响

Effect of CRKⅡon malignant behavior of chronic myeloid leukemia K562 cell

作为信号接头蛋白CRK家族成员之一,CRKⅡ参与细胞的增殖、存活、黏附、迁移、分化,但CRKⅡ与白血病的关系及作用机制尚不清楚。采用qRT-PCR(quantitative real-timePCR)检测CRKⅡ在正常人外周血样本、非恶性白血病患者骨髓样本、慢性粒细胞白血病(chronic myeloid leukemia,CML)初发患者骨髓样本、CML完全缓解(complete remission,CR)患者骨髓样本中的表达情况;CRKⅡ小干扰序列瞬时转染K562细胞,获得CRKⅡ表达下调的K562细胞株,Western blot和qRT-PCR检测CRKⅡ干扰效率;CCK8检测CRKⅡ下调对K562细胞增殖能力的影响;Transwell小室检测CRKⅡ下调对K562细胞迁移、侵袭能力的影响;Western blot检测CRKⅡ下调对K562细胞中信号通路关键分子p130Cas、Rac1表达水平的影响。与正常人外周血样本、非恶性白血病患者骨髓样本相比,CRKⅡ在CML初发患者骨髓样本中表达水平升高,同时在4对CML初发-CR配对样本中,患者完全缓解后CRKⅡ表达水平降低;在K562细胞中CRKⅡ表达被有效沉默;CRKⅡ下调抑制K562细胞的增殖、迁移、侵袭能力。初步分子机制研究发现,CRKⅡ通过激活p130Cas/DOCK180/Rac1通路调节K562细胞的恶性行为。CRKⅡ在CML发生、发展中起着重要的作用,作为肿瘤促进剂影响CML细胞的恶性行为,可作为CML诊断、治疗的新靶点。

As a member of CT10 regulation of kinase(CRK)adaptor protein family,CRKⅡis involved in cell proliferation,survival,adhesion,migration and differentiation.However,the exact function role and underlying mechanism of CRKⅡ in leukemia are still unclear.Quantitative real-time PCR(qRT-PCR)was used to detect the expression of CRKⅡ in normal peripheral blood(PB)samples,non-malignant leukemia patient bone marrow(BM)samples,chronic myeloid leukemia(CML)primary patient BM samples and CML complete remission(CR)patient BM samples.K562 cells were transiently transfected with small interference sequence of CRKⅡ to obtain K562 cells with CRKⅡ knockdown.Western blot and qRT-PCR were used to detect the interference efficiency of CRKⅡ.CCK8 assay was used to examine the effect of CRKⅡ downregulation on the proliferation ability of K562 cells.Transwell chamber assay was used to examine the effect of CRKⅡ downregulation on the migration and invasion abilities of K562 cells.Western blot was used to detect the expression levels of signaling pathway molecules p130 Cas and Rac1.CRKⅡ was overexpressed in CML primary patient BM samples compared with normal PB samples and non-malignant leukemia patient BM samples,meanwhile,in 4 pairs of CML primary-CR patient samples,the expression level of CRKⅡwas lower in CR patient samples than that in the corresponding CML primary patient samples.The expression of CRKⅡ was effectively silenced in K562 cells.CRKⅡ downregulation inhibited proliferation,migration and invasion abilities of K562 cells.Mechanistically,CRKⅡ regulated K562 cells malignant behavior via activating p130 Cas/DOCK180/Rac1 pathway.Taken together,our work demonstrates that CRKⅡ plays an important role in development and progression of CML and CRKⅡ,as a tumor promoter affects malignant behavior of CML cells,may be as a novel potential diagnosis and therapeutic target for CML.