DCLK1对结直肠癌细胞化疗敏感性的影响及机制

The effect and mechanism of DCLK1 on chemosensitivity of colorectal cancer cells

目的:探讨双肾上腺皮质激素样激酶1(DCLK1)对结直肠癌耐药细胞化疗敏感性的影响及机制。方法:采用实时定量PCR和Western blot方法检测人结直肠癌细胞系HT29、SW480及其奥沙利铂(OXA)耐药株HT29/OXA和SW480/OXA中DCLK1的表达。采用siRNA干扰HT29/OXA和SW480/OXA细胞中DCLK1的表达,并用不同浓度OXA干预细胞,CCK-8法检测细胞增殖及其对OXA的敏感性,Annexin V-FITC/PI双染法检测细胞凋亡,Caspase-3活性检测试剂盒检测细胞的Caspase-3活性,Western blot检测细胞中DCLK1、Bax、Bcl-2、多药耐药蛋白1(MDR1)、P-糖蛋白(P-gp)等蛋白的表达。结果:与亲本细胞系比较,DCLK1在HT29/OXA和SW480/OXA细胞中的mRNA和蛋白表达水平均显著上调(P<0.05)。转染DCLK1siRNA(si-DCLK1)后,HT29/OXA和SW480/OXA细胞的增殖活性均显著降低(P<0.05),且它们对OXA的敏感性显著增加(P<0.05);转染si-DCLK1后,HT29/OXA和SW480/OXA细胞的凋亡显著增加(P<0.05),伴随Caspase-3活性上调(P<0.05),Bax蛋白表达上调(P<0.05),Bcl-2蛋白表达下调(P<0.05);转染si-DCLK1后,HT29/OXA和SW480/OXA细胞中MDR1和P-gp的蛋白表达均显著下调(P<0.05)。结论:DCLK1可能通过调控Bax/Bcl-2以及MDR1和P-gp的表达影响结直肠癌细胞的化疗敏感性。

Objective:To investigate the effect of doublecortin-like kinase 1 (DCLK1) on chemosensitivity of colorectal cancer cells and its possible mechanism.Methods:Quantitative real-time PCR (qRT-PCR) and Western blot were performed to detect mRNA and protein expression of DCLK1 in human colorectal cancer cell lines HT29,SW480 and their oxaliplatin (OXA)-resistant cells HT29/OXA and SW480/OXA.The expression of DCLK1 in HT29/OXA and SW480/OXA cells was interfered with siRNA,and cells were treated with concentrations of OXA.Cell proliferation and their sensitivity to OXA were detected by CCK-8 assay.Cell apoptosis was detected by Annexin V-FITC/PI double staining assay.Caspase-3 activity assay kit was used to detect Caspase-3 activity,and Western blot was used to detect the protein expression of DCLK1,Bax,Bcl-2,multidrug resistance protein 1 (MDR1),and P-glycoprotein (P-gp).Results:Compared with the parental cell lines,the mRNA and protein expression of DCLK1 in HT29/OXA and SW480/OXA cells were significantly up-regulated ( P <0.05).After transfection of DCLK1 siRNA (si-DCLK1),the proliferation of HT29/OXA and SW480/OXA cells were significantly decreased ( P <0.05),and their sensitivity to OXA was significantly increased ( P <0.05).After si-DCLK1 transfection,the apoptosis of HT29/OXA and SW480/OXA cells was significantly increased ( P <0.05),along with the increased activity of Caspase-3 ( P <0.05),the up-regulation of Bax protein and down-regulation of Bcl-2 protein ( P <0.05).The protein expressions of MDR1 and P-gp were significantly down-regulated in si-DCLK1 transfected HT29/OXA and SW480/OXA cells ( P <0.05).Conclusion:DCLK1 may affect the chemosensitivity of colorectal cancer cells by regulating the expression of Bax/Bcl-2,MDR1 and P-gp.