Effect of DDX46 silencing on growth and apoptosis of esophageal carcinoma cells TE-1_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

 LI Bin ^1,2 , ZHU Duojie ¹ , HE Wenting ² , LIU Tao ² , WANG Cheng ¹ , SONG Tieniu ¹ , YANG Jianbao ¹ , GUO Quanwei ¹ , JIANG Peng ¹ , WEI Xiaoping ¹ , MENG Yuqi ¹

1. Department of Thoracic Surgery, Lanzhou University Second Hospital, Lanzhou University Second Clinical Medical College, Lanzhou, 730030, P.R.China;
2. Gansu Provincial Key Laboratory of Digestive System Tumors, Lanzhou, 730030, P.R.China;

Corresponding author：

LI Bin, Email: dr.leebin@outlook.com

Keywords：

Esophageal squamous cell carcinoma; DDX46; RNA interference; cell growth; cell apoptosis

DOI：

10.7507/1007-4848.201608084

Video：

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ObjectiveTo explore the effect of DDX46 silencing on growth and apoptosis in esophageal squamous cell carcinoma cell TE-1 by the shRNA. MethodsThe relative expression of DDX46 mRNA in TE-1 cells was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and compared with immortalized human esophageal squamous cell Het-1A. DDX46 shRNA-expressing lentivirus was applied to silence DDX46 (experimental group), and non-silencing control lentivirus was added (control group) with a multiplicity of infection of 5 in TE-1 cells. In both groups, cell growth was monitored using high content screening, cell colony-forming capacity was measured by colony formation assay, cell apoptosis were determined by flow cytometry. Further, the Stress and Apoptosis Signaling Antibody Array Kit was used to detect the changes of signaling molecules in TE-1 cells after DDX46 knockdown. ResultsCompared with the control group, cell counting after DDX46 silencing showed that TE-1 cell growth was significantly inhibited (P<0.001). Colony formation assay showed that cell colony-forming capacity was significantly inhibited (P<0.01). Annexin V-APC flow cytometry showed a significant increase in apoptosis (P<0.001). In PathScan® Antibody Array, the expression levels of Akt (Ser473, phosphorylation) and IκBα (Total, N/A) significantly decreased (P<0.01), and the expression of Caspase-3 (Asp175, cleaved) increased (P<0.05). ConclusionDDX46 is overexpressed in TE-1 cells. Targeted gene silencing of DDX46 inhibits cell growth, and induces cell apoptosis. DDX46 silencing probably by negative regulation of Akt/NF-κB signaling pathway, to play a role in inhibiting TE-1 cells growth and inducing apoptosis.

Citation： LI Bin, ZHU Duojie, HE Wenting, LIU Tao, WANG Cheng, SONG Tieniu, YANG Jianbao, GUO Quanwei, JIANG Peng, WEI Xiaoping, MENG Yuqi. Effect of DDX46 silencing on growth and apoptosis of esophageal carcinoma cells TE-1. Chinese Journal of Clinical Thoracic and Cardiovascular Surgery, 2017, 24(6): 463-468. doi: 10.7507/1007-4848.201608084 Copy

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13.	Abdelhaleem M. Do human RNA helicases have a role in cancer? Biochim Biophys Acta, 2004, 1704(1): 37-46.
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15.	Kosowski TR, Keys HR, Quan TK, et al. DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle. RNA, 2009, 15(7): 1345-1362.
16.	Liang WW, Cheng SC. A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence. Genes Dev, 2015, 29(1): 81-93.
17.	Abdelhaleem M, Maltais L, Wain H. The human DDX and DHX gene families of putative RNA helicases. Genomics, 2003, 81(6): 618-622.
18.	Li M, Ma YC, Huang P, et al. Lentiviral DDX46 knockdown inhibits growth and induces apoptosis in human colorectal cancer cells. Gene, 2015, 560(2): 237-244.
19.	Testa JR, Bellacosa A. AKT plays a central role in tumorigenesis. Proc Natl Acad Sci USA, 2001, 98(20): 10983-10985.
20.	田芳, 柴玉荣, 江亚南, 等. 姜黄素通过下调 IκBα 磷酸化抑制食管鳞癌细胞的体外增殖. 基础医学与临床, 2011, 31(7): 767-772.
21.	Gilmore TD. Introduction to NF-κB: players, pathways, perspectives. Oncogene, 2006, 25(51): 6680-6684.
22.	Ma CD, Zuo WS, Wang XW, et al. Lapatinib inhibits the activation of NF-κB through reducing phosphorylation of IκB-α in breast cancer cells. Oncol Rep, 2013, 29(2): 812-818.
23.	Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ, 1999, 6(2): 99-104.

1. Torre LA, Bray F, Siegel RL, et al. Global cancer statistics, 2012. CA Cancer J Clin, 2015, 65(2): 87-108.
2. Zheng RS, Zeng HM, Zhang SW, et al. National estimates of cancer prevalence in China, 2011. Cancer Lett, 2016, 370(1): 33-38.
3. Song YM, Li L, Ou YW, et al. Identification of genomic alterations in oesophageal squamous cell cancer. Nature, 2014, 509(7498): 91-95.
4. Linder P, Fuller-Pace FV. Looking back on the birth of DEAD-box RNA helicases. Biochim Biophys Acta, 2013, 1829(8): 750-755.
5. Robert F, Pelletier J. Perturbations of RNA helicases in cancer. Wiley Interdiscip Rev RNA, 2013, 4(4): 333-349.
6. Li B, Li YM, He WT, et al. Knockdown of DDX46 inhibits proliferation and induces apoptosis in esophageal squamous cell carcinoma cells. Oncol Rep, 2016, 36(1): 223-230.
7. Zielske SP, Stevenson M. Importin 7 may be dispensable for human immunodeficiency virus type 1 and simian immunodeficiency virus infection of primary macrophages. J Virol, 2005, 79(17): 11541-11546.
8. Fairman-Williams ME, Guenther UP, Jankowsky E. SF1 and SF2 helicases: family matters. Curr Opin Struct Biol, 2010, 20(3): 313-324.
9. Jarmoskaite I, Russell R. DEAD-box proteins as RNA helicases and chaperones. Wiley Interdiscip Rev RNA, 2011, 2(1): 135-152.
10. Owttrim GW. RNA helicases: diverse roles in prokaryotic response to abiotic stress. RNA Biol, 2013, 10(1): 96-110.
11. Hooper C, Hilliker A. Packing them up and dusting them off: RNA helicases and mRNA storage. Biochim Biophys Acta, 2013, 1829(8): 824-834.
12. Linder P, Lasko PF, Ashburner M, et al. Birth of the D-E-A-D box. Nature, 1989, 337(6203): 121-122.
13. Abdelhaleem M. Do human RNA helicases have a role in cancer? Biochim Biophys Acta, 2004, 1704(1): 37-46.
14. Xu YZ, Query CC. Competition between the ATPase Prp5 and branch region-U2 snRNA pairing modulates the fidelity of spliceosome assembly. Mol Cell, 2007, 28(5): 838-849.
15. Kosowski TR, Keys HR, Quan TK, et al. DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle. RNA, 2009, 15(7): 1345-1362.
16. Liang WW, Cheng SC. A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence. Genes Dev, 2015, 29(1): 81-93.
17. Abdelhaleem M, Maltais L, Wain H. The human DDX and DHX gene families of putative RNA helicases. Genomics, 2003, 81(6): 618-622.
18. Li M, Ma YC, Huang P, et al. Lentiviral DDX46 knockdown inhibits growth and induces apoptosis in human colorectal cancer cells. Gene, 2015, 560(2): 237-244.
19. Testa JR, Bellacosa A. AKT plays a central role in tumorigenesis. Proc Natl Acad Sci USA, 2001, 98(20): 10983-10985.
20. 田芳, 柴玉荣, 江亚南, 等. 姜黄素通过下调 IκBα 磷酸化抑制食管鳞癌细胞的体外增殖. 基础医学与临床, 2011, 31(7): 767-772.
21. Gilmore TD. Introduction to NF-κB: players, pathways, perspectives. Oncogene, 2006, 25(51): 6680-6684.
22. Ma CD, Zuo WS, Wang XW, et al. Lapatinib inhibits the activation of NF-κB through reducing phosphorylation of IκB-α in breast cancer cells. Oncol Rep, 2013, 29(2): 812-818.
23. Porter AG, Jänicke RU. Emerging roles of caspase-3 in apoptosis. Cell Death Differ, 1999, 6(2): 99-104.

Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Effect of DDX46 silencing on growth and apoptosis of esophageal carcinoma cells TE-1

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