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. |
[1] 张玥,石菊芳,黄慧瑶,等.中国人群结直肠癌疾病负担分析[J].中华流行病学杂志,2015,36(7):709-714.
[2] 王千里.结肠癌组织NPRL2表达及其临床意义研究[D].郑州:郑州大学,2014.
[3] 程雪,纪捷,张静敏,等.Lgr5蛋白和Her-2蛋白在结肠癌中的表达及其临床意义[J].现代医学,2017,45(1):17-22.
[4] 王勇,傅赞,封益飞,等.腹腔镜完整结肠系膜切除(CME)治疗局部晚期右半结肠癌的临床疗效[J].中国地方病防治杂志,2016(11):1302-1303.
[5] LIU A Y,LIU D G,DU Y J,et al.Relationship between tumor and peripheral blood NPRL2 mRNA levels in patients with colorectal adenoma and colorectal cancer[J].Cancer Biol Ther,2014,15(5):489-495.
[6] LIU A Y,LIU M N,PEI F H,et al.Functional characterization of the nitrogen permease regulator-like-2 candidate tumor suppressor gene in colorectal cancer cell lines[J].Mol Med Rep,2015,12(3):3487-3493.
[7] LIU M N,LIU A Y,DU Y J,et al.Nitrogen permease regulator-like 2 enhances sensitivity to oxaliplatin in colon cancer cells[J].Mol Med Rep,2015,12(1):1189-1196.
[8] LIU M,LIU A,PEI F,et al.Functional mechanism of the enhancement of 5-fluorouracil sensitivity by TUSC4 in colon cancer cells[J].Oncol Lett,2015,10(6):3682-3688.
[9] 向波,易梅,李小玲,等.细胞自噬在肿瘤发生发展中的作用[J].生物化学与生物物理进展,2012,39(3):45-53.
[10] 顾闻,何宋兵,汪良.自噬相关基因与结肠癌的研究进展[J].医学研究生学报,2014(4):414-417.
[11] LERMAN M I,MINNA J D.The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3:identification and evaluation of the resident candidate tumor suppressor genes[J].Cancer Res,2000,60(21):6116-6133.
[12] 刘虹,邵荣光.自噬在肿瘤发生与发展过程中的调节作用[J].药学学报,2016,51(1):23-28.
[13] TANIDA I,MINEMATSU-IKEGUCHI N,UENO T,et al.Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy[J].Autophagy,2005,1(2):84-91.
[14] BOYLE K B,RANDOW F.The role of ‘eat-me’ signals and autophagy cargo receptors in innate immunity[J].Curr Opin Microbiol,2013,16:339-348.
[15] 金镇勋,兰汝春,王菲,等.自噬抑制剂3-MA对乳胞素抑制胃癌BGC-823细胞增殖的影响及其机制[J].中国老年学,2012,32(5):977-978. |