DCLK1 对结直肠癌细胞化疗敏感性的影响及机制

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DCLK1 siRNA（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。

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

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.