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NPRL2对结肠癌HT29细胞系自噬和凋亡的影响
作者:刘艾芸  乔久涛  陈晶  裴凤华  杜雅菊 
单位:哈尔滨医科大学 附属第二医院, 黑龙江 哈尔滨 150081
关键词:人NPRL2 结肠肿瘤 自噬 凋亡 
分类号:R735.35
出版年·卷·期(页码):2019·38·第二期(249-255)
摘要:

目的:探讨NPRL2对人结肠癌细胞自噬的影响,并进一步探讨自噬和凋亡的关系。方法:构建过表达NPRL2慢病毒载体,转染至HT29细胞。检测转染48 h后NPRL2、LC3B和p62蛋白表达量,吖啶橙染色检测细胞自噬。用自噬抑制剂3-甲基腺嘌呤(3-MA)处理转染后细胞,测定细胞增殖,细胞死亡,细胞caspase-3活性,细胞凋亡和caspase-3、Bax、Bcl-2、LC3B及p62的蛋白表达量,吖啶橙染色检测细胞自噬。结果:免疫印迹法检测结果表明NPRL2能够激活HT29细胞的自噬。前期研究结果表明,NPRL2能够促进HT29细胞凋亡。进一步研究NPRL2激活的自噬与凋亡之间的关系,结果表明3-MA抑制由NPRL2诱导的自噬,降低细胞活力,抑制其增殖,促进其凋亡,并促进活化caspase-3和Bax的表达,抑制Bcl-2的蛋白表达和细胞自噬。结论:NPRL2能够促进HT29细胞的自噬,但这种自噬抑制了NPRL2引起的细胞凋亡,通过抑制3-MA抑制NPRL2引起的自噬能够有效地促进结肠癌细胞的凋亡。

Objective:To investigate the effects of nitrogen permease regulator-like-2 (NPRL2) on autophagy in human colon cancer HT29 cells, and further explore the relationship between autophagy and apoptosis. Methods:The lentiviral vector expressing the NPRL2 was constructed and transfected into HT29 cells. Western blot was used to detect the expressions of NPRL2, LC3B and p62 protein in HT29 cells after 48 h transfection and acridine orange staining were performed to observe the autophagy under a fluorescence microscope. The transfected HT29 cells were treated with autophagy inhibitor 3-methyladenine(3-MA), cell proliferation was measured by CCK-8, cell death was detected by Trypan blue, and caspase-3 activity was measured by caspase-3 activity kit. Cell apoptosis was measured by Annexin V-FITC/PI kit. Western blot was used to detect the protein expressions of active caspase-3, Bax, Bcl-2, LC3B and p62. Results:Western blot results showed that NPRL2 gene was successfully overexpressed in HT29 cells. Western blot results also revealed that NPRL2 can activate HT29 autophagy. And acridine orange staining were performed to observe the autophagy. Our published results showed that NPRL2 could promote the apoptosis of HT29 cells. To further investigate the relationship between NPRL2-activated autophagy and NPRL2-induced apoptosis, 3-MA was to treat used HT29 cells. The results showed that 3-MA inhibited the autophagy induced by NPRL2, and further reduced the cell viability of HT29 cell line, inhibited its proliferation, promoted its apoptosis, promoted the expressions of active caspase 3 and Bax, inhibited the expression of Bcl-2 and attenuated its autophagy. Conclusion:NPRL2 can promote the autophagy of colon cancer HT29 cell line, but this autophagy inhibits the apoptosis caused by NPRL2, and the inhibition of autophagy induced NPRL2 by 3-MA can effectively promote the apoptosis of colon cancer cells.

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