摘要
目的:研究甲氨蝶呤(methotrexate,MTX)耐药前后人大肠癌(结直肠癌)HT-29细胞中透明质酸蛋白聚糖连结蛋白1(hyaluronan and proteoglycan link protein 1,HAPLN1)表达的变化及其对MTX耐药性的影响,并探究可能的分子机制。方法:采用浓度梯度递增法构建耐药细胞株HT-29/MTX;RT-PCR检测HAPLN1和多药耐药相关蛋白2(multidrug resistance-associated protein 2,MRP2)的mRNA表达水平;采用脂质体介导法将人HAPLN1和MRP2基因的干扰质粒转染HT-29/MTX细胞并筛选出稳定表达的细胞系;采用CCK-8法检测细胞活力;流式细胞术检测细胞凋亡;采用Western blot检测HAPLN1、MRP2、IκB激酶(IκB kinase,IKK)α/β、p-IKKα/β(Ser176/Ser177)、p65和p-p65(Ser536)的蛋白水平。结果:HT-29/MTX细胞中HAPLN1和MRP2的mRNA和蛋白表达水平都显著高于其亲代HT-29细胞(P<0.05),耐药倍数高达463.756。抑制HAPLN1和MRP2基因表达使MTX对HT-29/MTX细胞的IC_(50)从15.304μmol/L分别降至6.119μmol/L和7.801μmol/L,逆转倍数分别为2.501和1.962,并增强MTX诱导的细胞凋亡(P<0.05)。敲减HAPLN1基因表达和使用IKK抑制剂IKK16均可下调IKKα/β和p65蛋白磷酸化水平以及MRP2蛋白表达水平(P<0.05),但IKK16未对HAPLN1蛋白表达产生影响(P>0.05)。结论:敲减HAPLN1基因表达可在体外增强人大肠癌耐药细胞株HT-29/MTX对MTX的敏感性,这可能与其抑制IKK/p65信号通路活化,继而下调MRP2基因表达有关。
AIM: To investigate the changes of hyaluronan and proteoglycan link protein 1(HAPLN1) expression before and after resistance to methotrexate(MTX) in human colorectal cancer HT-29 cells and its effect on this drug resistance, and to explore the molecular mechanism in the process. METHODS: The drug-resistant HT-29/MTX cells were established by stepwise exposure of the cells to MTX, and then the HT-29/MTX cells were stably transfected with specific shRNA interference plasmid vectors targeting HAPLN1 and multidrug resistance-associated protein 2(MRP2). The mRNA expression levels of HAPLN1 and MRP2 were measured by RT-PCR. CCK-8 assay was used to detect the viability of HT-29/MTX cells. The apoptosis rate was analyzed by flow cytometry. The protein levels of HAPLN1, MRP2, IκB kinase(IKK)α/β, p-IKKα/β(Ser176/Ser177), p65 and p-p65(Ser536) were determined by Western blot. RESULTS: The HT-29/MTX cells had significantly higher mRNA and protein levels of HAPLN1 and MRP2 than HT-29 cells(P<0.05) with resistant factor of 463.756. HAPLN1 and MRP2 gene silencing significantly increased the cytotoxicity and apoptosis of HT-29/MTX cells induced by MTX(P<0.05). The IC50 value was decreased from 15.304 μmol/L to 6.119 μmol/L and 7.801 μmol/L, respectively, and their reversal folds were 2.501 and 1.962, respectively. Silencing of HAPLN1 and IKK inhibitor IKK16 inhibited the phosphorylation of IKKα/β and p65(P<0.05), and down-regulated the protein level of MRP2 in the HT-29/MTX cells(P<0.05). However, IKK16 did not affect the protein level of HAPLN1 in the HT-29/MTX cells.CONCLUSION: Knock-down of HAPLN1 gene expression reverses the resistance to MTX in human colorectal cancer HT-29/MTX cells possibly by blocking the IKK/p65 signaling pathway and thus down-regulating the expression of MRP2.
作者
贺红柳
崔美英
王丰
冯晔子
袁磊
HE Hong-liu;CUI Mei-ying;WANG Feng;FENG Ye-zi;YUAN Lei(Department of Pathology, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052 , China;Department of Cardiovasology, Zhengzhou Seventh People’s Hospital, Zhengzhou 450000 , China;Key Laboratory of Medical Bioenginee-ring of Luohe City, Luohe Medical College, Luohe 462002 , China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2019年第4期621-627,共7页
Chinese Journal of Pathophysiology
基金
河南省科技厅科技发展计划项目(No.142102310203)
