Knockdown of estrogen receptor alpha inhibits the proliferation and migration of A549 cells and the formation of transplanted tumors in nude mice_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

 CHEN Peirui ¹ , LV Jing ¹ , ZENG Chunfang ² , HE Xiaoyan ³ ,  DIAO Mingqiang ¹

1. Department of Cardiothoracic Surgery, Deyang People’s Hospital, Deyang, Sichuan 618000, P. R. China;
2. Department of Respiratory Medicine, Deyang People’s Hospital, Deyang, Sichuan 618000, P. R. China;
3. Department of Pathology, Deyang People’s Hospital, Deyang, Sichuan 618000, P. R. China;

Corresponding author：

DIAO Mingqiang, Email: 1704981491@qq.com

Keywords：

Estrogen receptor α; A549 cells; Cell growth; Invasion; Tumor formation

DOI：

10.7507/1671-6205.202006060

Video：

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Objective To explore the effect of interfering RNA (shRNA) on biological activity of A549 cells and tumor growth in nude mice after knockdown of estrogen receptor α (ERα) gene. Methods The ERα gene in A549 cells was knocked down by shRNA. RT-PCR and Western blot were used to detect the gene expression and protein expression after knockdown; colony formation experiment was used to detect the proliferation of cells, and RT-PCR was used to detect the expression of Ki-67 and PCNA; flow cytometry was used to detect apoptosis rate; transwell assay was used to detect cell invasion ability; Western blot was used to detect the expression of epithelial cadherin (E-cad) and neuropathic cadherin (N-cad) protein. The control group and A549 cells transfected with ERα-shRNA1 were injected subcutaneously in nude mice to construct transplanted tumors. Immunohistochemistry was used to detect the expression of Ki-67 and N-cad in tumor tissues. Results Compared with the control group, after transfection of ERα-shRNA1 and ERα-shRNA2, the mRNA and protein expressions of ERα were reduced significantly (P<0.05), and shRNA1 with high interference efficiency was used for subsequent experiments. Compared with the control group, the A549 cells were transfected with ERα-shRNA1, the colony formation rate was down-regulated significantly (P<0.05), the apoptosis rate was increased significantly (P<0.05), the expression of Ki-67 and PCNA were down-regulated significantly (P<0.05), the number of invasive cells was reduced significantly, the expression of E-cad was increased, and the expression of N-cad was decreased (P<0.05). The results of tumor formation in nude mice showed that interfering with ERα expression can significantly inhibit tumor growth (P<0.05), and down-regulate the rate of Ki-67 and N-cad positive cells (P<0.05). Conclusion Knockdown of ERα inhibits the proliferation and migration ability of NSCLC cells and the occurrence and development of transplanted tumors in nude mice.

Citation： CHEN Peirui, LV Jing, ZENG Chunfang, HE Xiaoyan, DIAO Mingqiang. Knockdown of estrogen receptor alpha inhibits the proliferation and migration of A549 cells and the formation of transplanted tumors in nude mice. Chinese Journal of Respiratory and Critical Care Medicine, 2021, 20(9): 649-655. doi: 10.7507/1671-6205.202006060 Copy

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2.	Schwartz AG, Wenzlaff AS, Prysak GM, et al. Reproductive factors, hormone use, estrogen receptor expression and risk of non-small cell lung cancer in women. J Clin Oncol, 2007, 25(36): 5785‐5792.
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7.	Li Q, Gao H, Yang H, et al. Estradiol promotes the progression of ER+ breast cancer through methylation-mediated RSK4 inactivation. Onco Targets Ther, 2019, 12: 5907-5916.
8.	Che Q, Xiao X, Jun X, et al. 17beta-estradiol promotes endometrial cancer proliferation and invasion through IL-6 pathway. Endocr Connect, 2019, 8(7): 961-968.
9.	Frasor J, Danes JM, Komm B, et al. Estradiol promotes the progression up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology, 2003, 144(10): 4562-4574.
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12.	简红, 朱雷, 邵晋辰, 等. ERα、ERβ 在非吸烟 NSCLC 患者中的表达与生存期和性别的相关性研究. 中国癌症杂志, 2013, 23(11): 910-916.
13.	王伟, 郭雯, 董海北, 等. 灵芝孢子提取物对肺腺癌细胞顺铂化疗敏感性及 TAZ 表达的影响. 解放军医药杂志, 2020, 32(6): 19-23.
14.	杨丽莎, 李福祥. 沉默 CDH1 基因促进非小细胞肺癌 A549 细胞系转移侵袭的机制. 中国呼吸与危重监护杂志, 2019, 18(5): 452-456.
15.	吕薇, 龙波, 魏秀丽. miR-424 对肝癌细胞增殖、细胞周期和侵袭活性的影响. 解放军医药杂志, 2020, 32(4): 33-35.
16.	徐敬轩, 杨岩, 张勖, 等. 柴胡皂苷 D 通过下调 miR-517a 调节胶质瘤细胞体外和体内移植瘤生长. 医学分子生物学杂志, 2020, 17(1): 33-39.
17.	Tian W, Teng F, Gao J, et al. Estrogen and insulin synergistically promote endometrial cancer progression via crosstalk between their receptor signaling pathways. Cancer Biol Med, 2019, 16(1): 55-70.
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19.	许承斌, 丁明霞, 常媛媛, 等. 雌激素与非小细胞肺癌关系的研究进展. 医学综述, 2016, 22(21): 4215-4218.
20.	王紫翔, 白莎, 汪志国, 等. shRNA 干扰 lncRNA TUG1 对视网膜母细胞瘤生长的影响. 医学分子生物学杂志, 2020, 17(4): 285-292.
21.	Zhang J, Li G, Li Z, et al. Estrogen-independent effects of ERα36 in ER-negative breast cancer. Steroids, 2012, 77(6): 666-673.
22.	Zhang S, Qiu C, Wang L, et al. The elevated level of ERα36 is correlated with nodal metastasis and poor prognosis in lung adenocarcinoma. Steroids, 2014, 87: 39-45.
23.	Liu DD, Zhang CJ, Li XL, et al. MicroRNA-567 inhibits cell proliferation, migration and invasion by targeting FGF5 in osteosarcoma. EXCLI J, 2018, 17: 102-112.
24.	Tong JS, Zhang QH, Wang ZB, et al. ER-α36, a novel variant of ER-α, mediates estrogen-stimulated proliferation of endometrial carcinoma cells via the PKCδ/ERK pathway. PLoS One, 2010, 5(11): e15408.
25.	Reinbolt RE, Mangini N, Hill JL, et al. Endocrine therapy in breast cancer: the neoadjuvant, adjuvant, and metastatic approach. Semin Oncol Nurs, 2015, 31(2): 146-155.
26.	Liu YF, Ma H, Yao J. ERα, a key target for cancer therapy: a review. Onco Targets Ther, 2020, 13: 2183-2191.

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2016. CA Cancer J Clin, 2016, 66(1): 7‐30.
2. Schwartz AG, Wenzlaff AS, Prysak GM, et al. Reproductive factors, hormone use, estrogen receptor expression and risk of non-small cell lung cancer in women. J Clin Oncol, 2007, 25(36): 5785‐5792.
3. MarquezGarban DC, Chen HW, Goodglick L, et al. Targeting aromatase and estrogen signaling in human non‐small cell lung cancer. Ann N Y Acad Sci, 2009, 1155(1): 194‐205.
4. Zhang J, Guan X, Liang N, et al. Estrogen-related receptor alpha triggers the proliferation and migration of human non-small cell lung cancer via interleukin-6. Cell Biochem Funct, 2018, 36(5): 255-262.
5. Dahlman-Wright K, Cavailles V, Fuqua SA, et al. International union of pharmacology. LXIV. estrogen receptors. Pharmacol Rev, 2006, 58(4): 773-781.
6. Jameera Begam A, Jubie S, Nanjan MJ. Estrogen receptor agonists/antagonists in breast cancer therapy: a critical review. Bioorg Chem, 2017, 71: 257-274.
7. Li Q, Gao H, Yang H, et al. Estradiol promotes the progression of ER+ breast cancer through methylation-mediated RSK4 inactivation. Onco Targets Ther, 2019, 12: 5907-5916.
8. Che Q, Xiao X, Jun X, et al. 17beta-estradiol promotes endometrial cancer proliferation and invasion through IL-6 pathway. Endocr Connect, 2019, 8(7): 961-968.
9. Frasor J, Danes JM, Komm B, et al. Estradiol promotes the progression up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology, 2003, 144(10): 4562-4574.
10. 颜浩, 徐英. 非小细胞肺癌中两种雌激素受体亚型与血管内皮生长因子的表达及相关性研究. 中国呼吸与危重监护杂志, 2010, 9(3): 287-290.
11. Zhao G, Zhao S, Wang T, et al. Estrogen receptor β signaling regulates the progression of Chinese non-small cell lung cancer. J Steroid Biochem Mol Biol, 2011, 124(1-2): 47-57.
12. 简红, 朱雷, 邵晋辰, 等. ERα、ERβ 在非吸烟 NSCLC 患者中的表达与生存期和性别的相关性研究. 中国癌症杂志, 2013, 23(11): 910-916.
13. 王伟, 郭雯, 董海北, 等. 灵芝孢子提取物对肺腺癌细胞顺铂化疗敏感性及 TAZ 表达的影响. 解放军医药杂志, 2020, 32(6): 19-23.
14. 杨丽莎, 李福祥. 沉默 CDH1 基因促进非小细胞肺癌 A549 细胞系转移侵袭的机制. 中国呼吸与危重监护杂志, 2019, 18(5): 452-456.
15. 吕薇, 龙波, 魏秀丽. miR-424 对肝癌细胞增殖、细胞周期和侵袭活性的影响. 解放军医药杂志, 2020, 32(4): 33-35.
16. 徐敬轩, 杨岩, 张勖, 等. 柴胡皂苷 D 通过下调 miR-517a 调节胶质瘤细胞体外和体内移植瘤生长. 医学分子生物学杂志, 2020, 17(1): 33-39.
17. Tian W, Teng F, Gao J, et al. Estrogen and insulin synergistically promote endometrial cancer progression via crosstalk between their receptor signaling pathways. Cancer Biol Med, 2019, 16(1): 55-70.
18. Arun A, Ansari MI, Popli P, et al. New piperidine derivative DTPEP acts as dual-acting anti-breast cancer agent by targeting ERalpha and downregulating PI3K/Akt-PKCα leading to caspase-dependent apoptosis. Cell Prolif, 2018, 51(6): e12501.
19. 许承斌, 丁明霞, 常媛媛, 等. 雌激素与非小细胞肺癌关系的研究进展. 医学综述, 2016, 22(21): 4215-4218.
20. 王紫翔, 白莎, 汪志国, 等. shRNA 干扰 lncRNA TUG1 对视网膜母细胞瘤生长的影响. 医学分子生物学杂志, 2020, 17(4): 285-292.
21. Zhang J, Li G, Li Z, et al. Estrogen-independent effects of ERα36 in ER-negative breast cancer. Steroids, 2012, 77(6): 666-673.
22. Zhang S, Qiu C, Wang L, et al. The elevated level of ERα36 is correlated with nodal metastasis and poor prognosis in lung adenocarcinoma. Steroids, 2014, 87: 39-45.
23. Liu DD, Zhang CJ, Li XL, et al. MicroRNA-567 inhibits cell proliferation, migration and invasion by targeting FGF5 in osteosarcoma. EXCLI J, 2018, 17: 102-112.
24. Tong JS, Zhang QH, Wang ZB, et al. ER-α36, a novel variant of ER-α, mediates estrogen-stimulated proliferation of endometrial carcinoma cells via the PKCδ/ERK pathway. PLoS One, 2010, 5(11): e15408.
25. Reinbolt RE, Mangini N, Hill JL, et al. Endocrine therapy in breast cancer: the neoadjuvant, adjuvant, and metastatic approach. Semin Oncol Nurs, 2015, 31(2): 146-155.
26. Liu YF, Ma H, Yao J. ERα, a key target for cancer therapy: a review. Onco Targets Ther, 2020, 13: 2183-2191.

Chinese Journal of Respiratory and Critical Care Medicine

Knockdown of estrogen receptor alpha inhibits the proliferation and migration of A549 cells and the formation of transplanted tumors in nude mice

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