Atg7-siRNA通过调节精氨酸循环干扰食管癌ECA109细胞放疗敏感性

doi:10.6040/j.issn.1671-7554.0.2020.1295

摘要/Abstract

摘要： 目的探讨siRNA干扰自噬相关基因7(Atg7)后,对ECA109细胞放疗敏感性的影响以及对精氨酸循环相关通路的调控。方法食管癌ECA109细胞分为Ctrl组、siRNA组、Ctrl+R8Gy组(8Gy放射剂量处理的转染Atg7-Ctrl的ECA109细胞)和siRNA+R8Gy组(8Gy放射剂量处理的转染Atg7-siRNA的ECA109细胞);采用Western blotting法分别检测各组细胞Atg7、Beclin-1、微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)、精氨酸琥珀酸合成酶1(ASS1)和精氨酸琥珀酸裂解酶(ASL)蛋白的表达。CCK8实验检测精氨酸对ECA109细胞的影响,绘制不同浓度(0、8、16、32、64、128 mmol/L)精氨酸处理下的细胞存活曲线,并计算其IC50和IC10,将IC10作为后续实验的作用浓度。采用精氨酸和siRNA分别处理细胞,对细胞进行8Gy照射,24 h后通过计算细胞数量评估细胞的死亡率。结果无论放疗前后,siRNA干扰均可使细胞的Atg7蛋白下调(P<0.01)。与Ctrl组相比,Ctrl+R8Gy组自噬相关蛋白Beclin-1和LC3Ⅱ上调(P<0.01);与Ctrl+R8Gy组相比,siRNA+R8Gy组自噬相关蛋白Beclin-1和LC3Ⅱ下调(P<0.01)。与Ctrl+R8Gy组相比,siRNA+R8Gy组精氨酸循环相关通路蛋白ASS1和ASL上调(P<0.01)。精氨酸对ECA109细胞具有增殖抑制作用,且呈浓度和时间依赖性。放疗后24 h,细胞死亡量分析显示, siRNA干扰可以提高细胞死亡率(F=73.59,P<0.001),添加精氨酸可以提高细胞死亡率(F=158.72,P<0.001),siRNA和精氨酸同时作用时细胞死亡率最高(F=7.88,P=0.02)。结论放疗可以诱导ECA109细胞发生自噬,Atg7敲低可以拮抗放疗诱导的自噬,提高放疗诱导的ECA109细胞死亡率,增加ECA109细胞对放疗的敏感性。Atg7敲低可以调控ECA109细胞放疗后精氨酸循环相关通路蛋白,提高精氨酸浓度增加ECA109细胞的放射敏感性。

Abstract: Objective To investigate the effects of siRNA interference with autophagy related gene 7(Atg7)on the radiosensitivity of ECA109 cells and the regulation of arginine circulation-related pathways. Methods ECA109 cells were divided into four groups: Ctrl group, siRNA group, Ctrl+R8Gy group(ECA109 cells transfected with Atg7-Ctrl at 8Gy radiation dose)and siRNA+R8Gy group(ECA109 cells transfected with Atg7-siRNA at 8Gy radiation dose). The protein expressions of Atg7, Beclin-1, microtubule associated protein 1 light chain 3 Ⅱ(LC3 Ⅱ), arginine succinate synthetase1(ASS1)and arginine succinic acid lyase(ASL)in each group were detected with Western blotting. The effects of arginine were detected with CCK8 assay. The cell survival curves were drawn at different concentrations of arginine(0, 8, 16, 32, 64, 128 mmol/L). IC50 and IC10 were calculated, and IC10 was set as the action concentration in the subsequent experiments. Cells were treated with arginine and siRNA respectively, and irradiated with 8Gy. After 24 h, the cell mortality was assessed by counting the number of cells. Results siRNA interference down-regulated Atg7 before and after radiotherapy(P<0.01). Compared with the Ctrl group, the Ctrl+R8Gy group had up-regulated levels of Beclin-1 and LC3Ⅱ(P<0.01). Compared with the Ctrl+R8Gy group, the siRNA+R8Gy group had down-regulated levels of Beclin-1 and LC3Ⅱ(P<0.01). Compared with the Ctrl+R8Gy group, the siRNA+R8Gy group had up-regulated levels of ASS1 and ASL(P<0.01). Arginine inhibited the proliferation of ECA109 cells in a concentration and time-dependent manner. Cell death analysis showed that siRNA interference(F=73.59, P<0.001)and arginine addition(F=158.72, P<0.001)increased cell mortality. The cell mortality was at the highest level when siRNA and arginine were used in combination(F=7.88, P=0.02). Conclusion Radiotherapy can induce autophagy in ECA109 cells. The Atg7-siRNA interference effectively antagonizes the radiotherapy-induced increase in autophagy, increases the radiotherapy-induced ECA109 cell death, and enhances the radiosensitivity of ECA109 cells. Atg7 knockdown can regulate the arginine circulation-related pathway proteins of ECA109 cells after radiotherapy, and increasing arginine concentration can enhance radiosensitivity of ECA109 cells.

Key words: autophagy related gene 7-siRNA, Arginine succinate synthetase1, Arginine succinic acid lyase, Esophageal cancer, Arginine, Radiosensitivity

中图分类号:

R310.31

张小红,周云,杜秋莹,任慧欣,王超群. Atg7-siRNA通过调节精氨酸循环干扰食管癌ECA109细胞放疗敏感性[J]. 山东大学学报 (医学版), 2021, 59(4): 28-34.

ZHANG Xiaohong, ZHOU Yun, DU Qiuying, REN Huixin, WANG Chaoqun. Atg7-siRNA interferes with radiosensitivity of esophageal cancer ECA109 cells by regulating arginine circulation[J]. Journal of Shandong University (Health Sciences), 2021, 59(4): 28-34.

参考文献

[1] Huang P, Sun LY, Zhang YQ. A hopeful natural product, pristimerin, induces apoptosis, cell cycle arrest, and autophagy in esophageal cancer cells [J]. Anal Cell Pathol(Amst), 2019, 2019: 6127169. doi: 10.1155/2019/6127169.
[2] van Meerten E, van der Gaast A. Systemic treatment for oesophageal cancer [J]. Eur J Cancer, 2005, 41(5): 664-672.
[3] Ma H, Zheng S, Zhang X, et al. High mobility group box 1 promotes radioresistance in esophageal squamous cell carcinoma cell lines by modulating autophagy [J]. Cell Death Dis, 2019, 10(2): 136.
[4] Shi Y, Zhang B, Feng X, et al. Apoptosis and autophagy induced by DVDMs-PDT on human esophageal cancer Eca-109 cells [J]. Photodiagnosis Photodyn Ther, 2018, 24: 198-205. doi: 10.1016/j.pdpdt.2018.09.013.
[5] Sharma NK, Sarode SC, Sarode GS, et al. Starvation in cancer cells: circulating arginine is good for cancer but bad for patients [J]. Expert Rev Anticancer Ther, 2019, 19(6): 455-459.
[6] García-Navas R, Munder M, Mollinedo F. Depletion of L-arginine induces autophagy as a cytoprotective response to endoplasmic Reticulum stress in human T lymphocytes [J]. Autophagy, 2012, 8(11): 1557-1576.
[7] Zhang J, Pavlova NN, Thompson CB. Cancer cell metabolism: the essential role of the nonessential amino acid, glutamine [J]. EMBO J, 2017, 36(10): 1302-1315.
[8] Wu GY, Wu ZL, Dai ZL, et al. Dietary requirements of “nutritionally non-essential amino acids” by animals and humans [J]. Amino Acids, 2013, 44(4): 1107-1113.
[9] Shen LJ, Lin WC, Beloussow K, et al. Resistance to the anti-proliferative activity of recombinant arginine deiminase in cell culture correlates with the endogenous enzyme, argininosuccinate synthetase [J]. Cancer Lett, 2003, 191(2): 165-170.
[10] Shen LJ, Beloussow K, Shen WC. Modulation of arginine metabolic pathways as the potential anti-tumor mechanism of recombinant arginine deiminase [J]. Cancer Lett, 2006, 231(1): 30-35.
[11] Feun LG, Kuo MT, Savaraj N. Arginine deprivation in cancer therapy[J]. Curr Opin Clin Nutr Metab Care, 2015, 18(1): 78-82.
[12] Patil MD, Bhaumik J, Babykutty S, et al. Arginine dependence of tumor cells: targeting a chink in cancers armor [J]. Oncogene, 2016, 35(38): 4957-4972.
[13] Cao Y, Feng Y, Zhang Y, et al. L-Arginine supplementation inhibits the growth of breast cancer by enhancing innate and adaptive immune responses mediated by suppression of MDSCs in vivo [J]. BMC Cancer, 2016, 16: 343. doi: 10.1186/s12885-016-2376-0.
[14] Jahani M, Azadbakht M, Rasouli H, et al. L-arginine/5-fluorouracil combination treatment approaches cells selectively: Rescuing endothelial cells while killing MDA-MB-468 breast cancer cells [J]. Food Chem Toxicol, 2019, 123: 399-411. doi: 10.1016/j.fct.2018.11.018.
[15] Geiger R, Rieckmann JC, Wolf T, et al. L-arginine modulates T cell metabolism and enhances survival and anti-tumor activity [J]. Cell, 2016, 167(3): 829-842.
[16] Bahl R, Arora S, Nath N, et al. Novel polymorphism in p21(waf1/cip1)cyclin dependent kinase inhibitor gene: association with human esophageal cancer [J]. Oncogene, 2000, 19(3): 323-328.
[17] 章海容, 张小红, 王超群. 哺乳动物雷帕霉素靶蛋白通路调控ECA109细胞放疗敏感性的代谢组学[J]. 山东大学学报(医学版), 2020, 58(1): 6-12. ZHANG Hairong, ZHANG Xiaohong, WANG Chaoqun. A metabonomic study on mTOR pathway regulating radiosensitivity of ECA109 cells[J]. Journal of Shandong University(Health Sciences), 2020, 58(1): 6-12.
[18] Lum JJ, Bauer DE, Kong M, et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis [J]. Cell, 2005, 120(2): 237-248.
[19] Apel A, Herr I, Schwarz H, et al. Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy [J]. Cancer Res, 2008, 68(5): 1485-1494.
[20] Benzina S, Altmeyer A, Malek F, et al. High-LET radiation combined with oxaliplatin induce autophagy in U-87 glioblastoma cells [J]. Cancer Lett, 2008, 264(1): 63-70.
[21] Brech A, Ahlquist T, Lothe RA, et al. Autophagy in tumour suppression and promotion [J]. Mol Oncol, 2009, 3(4): 366-375.
[22] Chen YS, Song HX, Lu Y, et al. Autophagy inhibition contributes to radiation sensitization of esophageal squamous carcinoma cells [J]. Dis Esophagus, 2011, 24(6): 437-443.
[23] 陆怡, 濮丽英, 周利群, 等. 免疫营养在恶性肿瘤治疗中的研究现状及进展[J]. 现代肿瘤医学, 2017, 25(22): 3722-3726. LU Yi, PU Liying, ZHOU Liqun, et al. Present status and progress of immunonutrition in the treatment of malignant tumor [J]. Journal of Modern Oncology, 2017, 25(22): 3722-3726.
[24] Lee HY, Mohammed KA, Goldberg EP, et al. Arginine-conjugated albumin microspheres inhibits proliferation and migration in lung cancer cells [J]. Am J Cancer Res, 2013, 3(3): 266-277.
[25] Li X, Wenes M, Romero P, et al. Navigating metabolic pathways to enhance antitumour immunity and immunotherapy [J]. Nat Rev Clin Oncol, 2019, 16(7): 425-441.
[26] Stasyk OV, Boretsky YR, Gonchar MV, et al. Recombinant arginine-degrading enzymes in metabolic anticancer therapy and bioanalytics [J]. Cell Biol Int, 2015, 39(3): 246-252.
[27] Kuo MT, Savaraj N, Feun LG. Targeted cellular metabolism for cancer chemotherapy with recombinant arginine-degrading enzymes [J]. Oncotarget, 2010, 1(4): 246-251.
[28] Li Y, Zeng X, Wang S, et al. Blocking autophagy enhanced leukemia cell death induced by recombinant human arginase [J]. Tumour Biol, 2016, 37(5): 6627-6635.

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed