摘要
目的探究MutT同源蛋白1(MutT homolog protein 1,MTH1)在结肠癌组织中的表达情况,以及下调MTH1对人结肠癌细胞SW480安罗替尼耐药的作用及其与自噬的关系。方法免疫组化染色法和实时荧光定量PCR(qRT-PCR)检测结肠癌组织和癌旁组织中MTH1表达;通过低剂量反复刺激法构建安罗替尼的SW480耐药细胞株(SW480/Anl细胞);利用RNA干扰技术下调SW480/Anl细胞中MTH1表达;CCK-8法检测细胞增殖能力;Annexin V-FITC/PI双染法测定细胞凋亡率;免疫荧光染色检测细胞中LC-3表达;蛋白质印迹法(Western blotting)检测MTH1蛋白及自噬蛋白表达水平。结果结肠癌组织中MTH1表达水平较癌旁组织显著升高(P<0.05);经不同浓度安罗替尼处理后,SW480/Anl细胞存活率较SW480细胞显著升高(P<0.05)。与对照组比较,转染siRNA-MTH1后SW480/Anl细胞中MTH1 mRNA与蛋白表达显著下降(P<0.05),在安罗替尼(浓度为25、50、100、150、200μmol/L)作用下细胞存活率均显著下降(P<0.05),细胞凋亡率升高(P<0.05),LC3荧光染色较弱,P62蛋白表达水平显著增加,同时LC3-Ⅱ、Beclin-1蛋白表达水平显著下降(P<0.05)。结论MTH1在结肠癌组织中高表达,下调MTH1表达可抑制自噬的发生,从而逆转SW480细胞对安罗替尼的耐药。
Objective To investigate the expression of MutT homolog protein 1(MTH1)in colon cancer tissues,and to down-regulate the resistance of MTH1 to human colon cancer cell line SW480 Anlotinib and its relationship with autophagy relationship.Methods Immunohistochemical staining and real-time fluorescence quantitative PCR(qRT-PCR)were used to detect the expression of MTH1 in colon cancer tissues and adjacent tissues.Low-dose repeated stimulation method was used to construct SW480 Anlotinib resistant cell line(SW480/Anl cells).RNA interference technology was used to down-regulate the expression of MTH1 in SW480/Anl cells;CCK-8 method was used to detect the cell proliferation ability;Annexin V-FITC/PI double staining method was used to determine the apoptosis rate;immunofluorescence staining was used to detect the expression of LC-3 in cells;Western blotting was used to detect the expression levels of MTH1 protein and autophagy protein.Results The expression level of MTH1 in colon cancer tissue was significantly higher than that in adjacent tissues(P<0.05);after treatment with different concentrations of Anlotinib,the survival rate of SW480/Anl cells was significantly higher than that of SW480 cells(P<0.05).Compared with the control group,the expression of MTH1 mRNA and protein in SW480/Anl cells decreased significantly after transfection of siRNA-MTH1(P<0.05).Under the action of Anlotinib(at the concentration of 25,50,100,150,200μmol/L),the cell survival rate decreased significantly(P<0.05),the apoptosis rate increased(P<0.05),LC3 fluorescence staining was weak,the expression level of P62 protein increased significantly,and the expression levels of LC3-Ⅱand Beclin-1 protein decreased significantly(P<0.05).Conclusion The high expression of MTH1 in colon cancer tissues,down-regulation of MTH1 expression can inhibit the occurrence of autophagy,thereby reversing the resistance of SW480 cells to Anlotinib.
作者
程伟
胡军
周琴
高丽霞
冀红红
朱海鹏
张益枝
木拉格尔·孜克力亚
张斌
CHENG Wei;HU Jun;ZHOU Qin;GAO Lixia;JI Honghong;ZHU Haipeng;ZHANG Yizhi;Mulageer·ZIKELIYA;ZHANG Bin(Department of Hematology and Oncology,Karamay Central Hospital,Karamay 834000,China)
出处
《胃肠病学和肝病学杂志》
CAS
2021年第8期868-873,878,共7页
Chinese Journal of Gastroenterology and Hepatology
基金
白求恩·医学科学研究基金资助项目(B19293BT)。
