外源性AGR2对结肠癌细胞增殖及侵袭能力的影响

doi:10.3760/cma.j.cn371439-20210526-00011

摘要/Abstract

摘要：

目的检测3种结肠癌细胞株SW480、SW620和COLO205培养液中前梯度蛋白2(AGR2)的表达情况,并探讨不同浓度外源性AGR2对SW620细胞增殖、迁移和侵袭能力的影响。方法采用蛋白质印迹法检测结肠癌细胞SW480、SW620和COLO205培养液中AGR2蛋白表达水平。将SW620细胞分为空白对照组、前梯度蛋白2同源人重组蛋白(rAGR2)低浓度组(100 μg/ml)和rAGR2高浓度组(200 μg/ml),采用CCK-8实验、细胞划痕实验、Transwell迁移和侵袭实验检测不同浓度rAGR2对SW620细胞生物学行为的影响。结果蛋白质印迹法检测结果显示,SW480、SW620、COLO205细胞AGR2蛋白表达水平分别为0.545±0.097、0.662±0.040、0.882±0.156,差异有统计学意义(F=7.46,P=0.024),COLO205细胞株AGR2蛋白水平明显高于SW480、SW620细胞株(P=0.009;P=0.047)。CCK-8实验结果显示,空白对照组、rAGR2低浓度组、rAGR2高浓度组SW620细胞增殖活性分别为0.422±0.031、0.542±0.040、0.574±0.033,差异有统计学意义(F=26.35,P<0.001),rAGR2低浓度组、rAGR2高浓度组明显高于空白对照组(均P<0.001)。细胞划痕实验结果显示,3组细胞36 h细胞划痕面积百分比分别为(28.029±2.107)%、(20.642±0.983)%、(16.951±1.608)%,差异有统计学意义(F=35.85,P<0.001),rAGR2低浓度组高于空白对照组(P=0.001),rAGR2高浓度组高于rAGR2低浓度组(P=0.032)。细胞迁移实验结果显示,3组细胞迁移数分别为(447.1±32.3)个、(513.1±55.8)个、(632.4±50.3)个,差异有统计学意义(F=35.62,P<0.001),rAGR2低浓度组多于空白对照组(P=0.007),rAGR2高浓度组多于rAGR2低浓度组(P<0.001)。侵袭实验结果显示,3组细胞侵袭数分别为(369.1±56.1)个、(505.1±34.4)个、(579.0±71.5)个,差异有统计学意义(F=32.40,P<0.001),rAGR2低浓度组多于空白对照组(P<0.001),rAGR2高浓度组多于rAGR2低浓度组(P=0.010)。结论不同侵袭性结肠癌细胞的细胞外液中AGR2蛋白表达量不同,随着侵袭能力的增高,AGR2蛋白的表达也增高。AGR2蛋白可提高结肠癌细胞SW620增殖、迁移和侵袭能力。

关键词: 结肠肿瘤, 细胞增殖, 细胞运动, AGR2

Abstract:

Objective To detect the expressions of anterior gradient protein 2 (AGR2) in the cultures of three colon cancer cell lines SW480, SW620 and COLO205, and to investigate the effects of different concentrations of exogenous AGR2 on the proliferation, migration and invasion abilities of SW620 cells. Methods Western blotting was used to detect the expression levels of AGR2 protein in SW480, SW620 and COLO205 colon cancer cell cultures. SW620 cells were divided into blank control group, anterior gradient protein 2 homologous human recombinant protein (rAGR2) low concentration group (100 μg/ml) and rAGR2 high concentration group (200 μg/ml), and CCK-8 assay, cell scratch assay and Transwell migration and invasion assay were used to detect the effects of different concentrations of rAGR2 on the biological behaviors of SW620 cells. Results Western blotting results showed that the expression levels of AGR2 protein in SW480, SW620 and COLO205 cells were 0.545±0.097, 0.662±0.040 and 0.882±0.156 respectively, with a statistically significant difference (F=7.46, P=0.024). The level of AGR2 protein in COLO205 cell line was significantly higher than that in SW480 and SW620 cell lines (P=0.009; P=0.047). The results of CCK-8 experiment showed that the proliferative activities of SW620 cells in the blank control group, rAGR2 low concentration group and rAGR2 high concentration group were 0.422±0.031, 0.542±0.040 and 0.574±0.033 respectively, with a statistically significant difference (F=26.35, P<0.001), and the rAGR2 low concentration group and rAGR2 high concentration group were significantly higher than the blank control group (both P<0.001). The results of cell scratching assay showed that the percentage of 36 h cell scratching area was (28.029±2.107)%, (20.642±0.983)% and (16.951±1.608)% for the three groups of cells respectively, with a statistically significant difference (F=35.85, P<0.001), the rAGR2 low concentration group was higher than the blank control group (P=0.001), and the rAGR2 high concentration group was higher than the rAGR2 low concentration group (P=0.032). The results of cell migration assay showed that the number of cells migrated in the three groups was 447.1±32.3, 513.1±55.8 and 632.4±50.3 respectively, with a statistically significant difference (F=35.62, P<0.001), the rAGR2 low concentration group was more than the blank control group (P=0.007), and the rAGR2 high concentration group was more than the rAGR2 low concentration group (P<0.001). The results of the invasion assay showed that the number of cells invaded in the three groups was 369.1±56.1, 505.1±34.4 and 579.0±71.5 respectively, with a statistically significant difference (F=32.40, P<0.001), the rAGR2 low concentration group was more than the blank control group (P<0.001), and the rAGR2 high concentration group was more than the rAGR2 low concentration group (P=0.010). Conclusion The expression of AGR2 protein varies in the extracellular fluid of different invasive colon cancer cells and increases with the invasive ability. AGR2 protein can increase the proliferation, migration and invasive abilities of colon cancer cells SW620.

Key words: Colonic neoplasms, Cell proliferation, Cell movement, AGR2

来比江·吾斯曼, 曹博威, 张文斌, 高华. 外源性AGR2对结肠癌细胞增殖及侵袭能力的影响[J]. 国际肿瘤学杂志, 2022, 49(2): 73-78.

Laibijiang Wusiman, Cao Bowei, Zhang Wenbin, Gao Hua. Effects of exogenous AGR2 on the proliferation and invasion abilities of colon cancer cells[J]. Journal of International Oncology, 2022, 49(2): 73-78.

图/表 3

参考文献 11

[1]	Chevet E, Fessart D, Delom F, et al. Emerging roles for the pro-oncogenic anterior gradient-2 in cancer development[J]. Oncogene, 2013, 32(20): 2499-2509. DOI: 10.1038/onc.2012.346. doi: 10.1038/onc.2012.346 pmid: 22945652
[2]	Moidu NA, A Rahman NS, Syafruddin SE, et al. Secretion of pro-oncogenic AGR2 protein in cancer[J]. Heliyon, 2020, 6(9): e05000. DOI: 10.1016/j.heliyon.2020.e05000. doi: 10.1016/j.heliyon.2020.e05000
[3]	Li Y, Wang W, Liu Z, et al. AGR2 diagnostic value in nasopharyngeal carcinoma prognosis[J]. Clin Chim Acta, 2018, 484:323-327. DOI: 10.1016/j.cca.2017.12.023. doi: 10.1016/j.cca.2017.12.023
[4]	王钧, 李蔚范. 宫颈癌患者手术前后血清及病变组织AGR2水平变化及其临床意义[J]. 西部医学, 2018, 30(5): 697-700. DOI: 10.3969/j.issn.1672-3511.2018.05.017. doi: 10.3969/j.issn.1672-3511.2018.05.017
[5]	Kereh DS, Pieter J, Hamdani W, et al. Correlation of AGR2 expression with the incidence of metastasis in luminal breast cancer[J]. Breast Dis, 2021, 40(S1): S103-S107. DOI: 10.3233/BD-219015. doi: 10.3233/BD-219015
[6]	Shi T, Gao Y, Quek SI, et al. A highly sensitive targeted mass spectrometric assay for quantification of AGR2 protein in human urine and serum[J]. J Proteome Res, 2014, 13(2): 875-882. DOI: 10.1021/pr400912c. doi: 10.1021/pr400912c
[7]	刘建捷, 高华, 许新才, 等. 结肠癌中AGR2和S100A4的表达及临床意义[J]. 临床与实验病理学杂志, 2018, 34(4): 436-439. DOI: 10.13315/j.cnki.cjcep.2018.04.018. doi: 10.13315/j.cnki.cjcep.2018.04.018
[8]	Tian S, Hu J, Tao K, et al. Secreted AGR2 promotes invasion of colorectal cancer cells via Wnt11-mediated non-canonical Wnt signaling[J]. Exp Cell Res, 2018, 364(2): 198-207. DOI: 10.1016/j.yexcr.2018.02.004. doi: 10.1016/j.yexcr.2018.02.004
[9]	Xue X, Fei X, Hou W, et al. miR-342-3p suppresses cell proliferation and migration by targeting AGR2 in non-small cell lung cancer[J]. Cancer Lett, 2018, 412:170-178. DOI: 10.1016/j.canlet.2017.10.024. doi: 10.1016/j.canlet.2017.10.024
[10]	Guo H, Zhu Q, Yu X, et al. Tumor-secreted anterior gradient-2 binds to VEGF and FGF2 and enhances their activities by promoting their homodimerization[J]. Oncogene, 2017, 36(36): 5098-5109. DOI: 10.1038/onc.2017.132. doi: 10.1038/onc.2017.132 pmid: 28481872
[11]	于天弘, Merugu SB, Negi H, et al. 抗AGR2单克隆抗体18A4抑制细胞外AGR2诱导的肿瘤细胞活性[J]. 中国药科大学学报, 2018, 49(2): 238-246. DOI: 10.11665/j.issn.1000-5048.20180215. doi: 10.11665/j.issn.1000-5048.20180215

组别
空白对照组	55.457±0.743	45.597±0.453	37.362±1.081	28.029±2.107
rAGR2低浓度组	53.666±2.158	46.686±1.047	30.745±1.709^a	20.642±0.983^a
rAGR2高浓度组	53.876±0.325	46.142±0.594	28.864±0.737^a	16.951±1.608^ab
F值	1.62	1.61	38.70	35.85
P值	0.273	0.275	<0.001	<0.001

[1]	任露, 谢晓丽, 张坤, 王丽娟. 双氢青蒿素联合卡非佐米对多发性骨髓瘤细胞活性、增殖、凋亡的影响及机制研究[J]. 国际肿瘤学杂志, 2024, 51(3): 129-136.
[2]	向玉玲, 谭佳杰, 熊远果, 赵丽蓉, 黎晨, 张洪. 脱水淫羊藿素对肝癌细胞增殖、迁移和凋亡的影响[J]. 国际肿瘤学杂志, 2023, 50(9): 513-519.
[3]	全祯豪, 徐飞鹏, 黄哲, 黄先进, 陈日红, 孙开裕, 胡旭, 林琳. 沉默lncRNA FTX通过miR-22-3p/NLRP3炎症体通路抑制胃癌细胞增殖[J]. 国际肿瘤学杂志, 2023, 50(4): 202-207.
[4]	褚雪镭, 毛昀, 薛鹏, 李林潞, 陈美池, 袁淳晟, 秦晓艳, 朱世杰. 化疗剂量强度对晚期结肠癌患者近期疗效的影响：基于真实世界数据研究[J]. 国际肿瘤学杂志, 2022, 49(7): 408-415.
[5]	连海伟, 杨烁锐, 刘仁忠. 金松双黄酮联合CX-4945通过Notch1通路调控胶质母细胞瘤细胞增殖与凋亡的机制研究[J]. 国际肿瘤学杂志, 2022, 49(6): 321-326.
[6]	周仁邦, 张仲传, 许志远, 朱勋兵. miR-219a-5p通过负调控HMGA2抑制骨肉瘤U2OS细胞增殖、侵袭和迁移[J]. 国际肿瘤学杂志, 2022, 49(4): 193-198.
[7]	石雨, 陈曦, 许梦琪, 张滢滢, 冀洪海, 蒋英英. 沉默PSIP1基因对口腔鳞状细胞癌细胞迁移及侵袭的影响[J]. 国际肿瘤学杂志, 2022, 49(3): 129-133.
[8]	张永丽, 张若佳, 范焕彩, 葛鲁娜, 王林. TXNDC5-Prx2途径对前列腺癌细胞耐药性的调控[J]. 国际肿瘤学杂志, 2021, 48(8): 473-478.
[9]	蒋梦婷, 王家淳, 宗静. 抑制NFAT5对肺腺癌细胞增殖、侵袭、迁移及凋亡能力的影响[J]. 国际肿瘤学杂志, 2021, 48(6): 321-327.
[10]	阎星羽, 廉振颖, 刁玉涛, 刘红艳. BMXΔN通过ERK/MAPK信号通路影响肺癌细胞对吉非替尼的耐药性[J]. 国际肿瘤学杂志, 2021, 48(6): 328-334.
[11]	王子宜, 陈鸿杰, 杨宁刚, 张骏, 张向军, 于新宁, 马忠义, 戴恩来. 核心蛋白聚糖对膀胱癌细胞增殖、迁移及侵袭的影响[J]. 国际肿瘤学杂志, 2021, 48(6): 335-340.
[12]	马秀珍, 卢艳, 赵冰冰, 邱宏聪, 徐勋, 韦敏. 岗松总黄酮对宫颈癌SiHa细胞迁移、侵袭及凋亡的影响[J]. 国际肿瘤学杂志, 2021, 48(4): 206-211.
[13]	李倩倩, 朱晓娣, 赵荣兰, 彭效祥. P2X7受体在结肠癌进展中的作用[J]. 国际肿瘤学杂志, 2021, 48(4): 250-253.
[14]	李炳亮, 杨娅, 黄英丽, 司文, 李兴伟, 张元民, 卞继超, 陈语. miR-20a-5p靶向KDM6B对骨肉瘤细胞增殖、迁移和侵袭能力的影响[J]. 国际肿瘤学杂志, 2021, 48(2): 65-73.
[15]	赵丽丽, 赵文文, 冯青青, 赵文飞, 张雪, 井文君, 魏红梅. 沉默PD-L1表达对胃癌细胞生物学行为的影响[J]. 国际肿瘤学杂志, 2021, 48(12): 705-710.