Int J Biol Sci 2020; 16(6):1010-1022. doi:10.7150/ijbs.40173

Research Paper

Knockdown of SMAD3 inhibits the growth and enhances the radiosensitivity of lung adenocarcinoma via p21 in vitro and in vivo

Hao Niu1*, Yiwei Huang2*, Li Yan3, Li Zhang1, Mengnan Zhao2, Tao Lu2, Xiaodong Yang2, Zhengcong Chen2, Cheng Zhan2✉, Yu Shi2✉, Qun Wang2

1. Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai, China.
2. Department of Thoracic Surgery, Zhongshan Hospital, Fudan University, Shanghai, China.
3. Department of Radiation Oncology, Eye & ENT Hospital, Fudan University, Shanghai, China.
*Hao Niu and Yiwei Huang contributed equally to this work.

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Citation:
Niu H, Huang Y, Yan L, Zhang L, Zhao M, Lu T, Yang X, Chen Z, Zhan C, Shi Y, Wang Q. Knockdown of SMAD3 inhibits the growth and enhances the radiosensitivity of lung adenocarcinoma via p21 in vitro and in vivo. Int J Biol Sci 2020; 16(6):1010-1022. doi:10.7150/ijbs.40173. Available from http://www.ijbs.com/v16p1010.htm

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Abstract

Radiotherapy is an effective approach for the treatment of lung adenocarcinoma. However, evidence suggests that lung adenocarcinoma can easily develop tolerance to radiotherapy. The purpose of this study was to investigate the effect and mechanism of SMAD3 on the radiosensitivity of lung adenocarcinoma in vitro and in vivo. We found that knockdown of SMAD3 using two short hairpin RNAs in lentivirus vectors significantly inhibited cell growth and increased radiosensitivity of the lung adenocarcinoma cell lines A549, H1299, and H1975. Using RNA sequencing and bioinformatics analyses, we found that the significantly differentially expressed genes in SMAD3 knockdown cells were mainly enriched in the cell cycle process. We then showed that knockdown of SMAD3 significantly reduced expression of cyclin-dependent kinase inhibitor 1 (p21) and increased the proportion of G2/M phase cells and the radiosensitivity of lung adenocarcinoma. Chromatin immunoprecipitation results in the Gene Expression Omnibus (GEO) database and our luciferase assay verified that SMAD3 directly bound the p21 promoter. A series of rescue experiments showed that overexpression of p21 partly reversed the effect of SMAD3 on proliferation and radioresistance in vitro and in vivo. Moreover, we found that the expression levels of SMAD3 and p21 were highly correlated, and both correlated with the patients' survival in online databases and clinical specimens. Expression of SMAD3 and p21 was also significantly different between radioresistant and radiosensitive patients in our hospital. Our results indicate that SMAD3 is a potential prognosis and radiosensitivity indicator as well as a target for radiotherapy and other treatments of patients with lung adenocarcinoma.

Keywords: SMAD3, radio-sensitivity, lung adenocarcinoma, cell cycle, p21, prognosis