Effect of DUS4L knockdown on gene expression regulation of human A549 lung adenocarcinoma cell line and analysis of different genes_Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Authors：

LI Jie , LI Zheng ,  LI Bin , YU Qiyao , MAO Wenjie , MENG Yuqi , ZHU Duojie , FENG Haiming , YIN Ci , XIAO Cui

Department of Thoracic Surgery, Lanzhou University Second Hospital, Lanzhou University Second Clinical Medical College, Lanzhou, 730030, P. R. China;

Corresponding author：

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

Keywords：

Dihydrouridine synthase 4-like; lung adenocarcinoma; RNA-seq; differential genes; bioinformatics analysis

DOI：

10.7507/1007-4848.202102059

Video：

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Objective To explore the mechanism of dihydrouridine synthase 4-like (DUS4L) on the development of lung adenocarcinoma (LUAD).Methods The RNA-seq expression data of LUAD was downloaded from The Cancer Genome Atlas (TCGA), and the relationship between its clinical pathological characteristics and DUS4L mRNA expression was evaluated. The effect of DUS4L knockdown on the proliferation of A549 cells was detected by EDU proliferation assay. The gene expression profile of lung adenocarcinoma A549 cells in the DUS4L knockdown group (KD group) and control group (NC group) was detected by transcriptome sequencing technique. The differential genes were screened by DESeq2. ClusterProfiler was used to perform GO functional enrichment analysis of differential genes.Results The expression of DUS4L mRNA in LUAD tissues was higher than that in normal tissues, and the up-regulation of DUS4L was related to the clinical pathological characteristics of LUAD patients. EDU proliferation assay suggested that knocking down DUS4L could inhibit the proliferation of A549 cells. A total of 456 differential genes were screened, including 289 up-regulated genes and 167 down-regulated genes [|log₂(fold change)|>1 and P_adj<0.05]. STC2 and TRIB3 were significantly down-regulated (P<0.05). Differential genes were mainly involved in the production of interleukin-8, angiogenesis, vascular endothelial cell proliferation and other biological pathways.Conclusion DUS4L can widely regulate the gene expression of LUAD cells, which provides a new idea for further studying the function and role of DUS4L in the occurrence and development of LUAD and finding new therapeutic targets for LUAD.

Citation： LI Jie, LI Zheng, LI Bin, YU Qiyao, MAO Wenjie, MENG Yuqi, ZHU Duojie, FENG Haiming, YIN Ci, XIAO Cui. Effect of DUS4L knockdown on gene expression regulation of human A549 lung adenocarcinoma cell line and analysis of different genes. Chinese Journal of Clinical Thoracic and Cardiovascular Surgery, 2022, 29(6): 761-769. doi: 10.7507/1007-4848.202102059 Copy

1.	Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2021, 71(3): 209-249.
2.	Yatabe Y, Borczuk AC, Powell CA. Do all lung adenocarcinomas follow a stepwise progression? Lung Cancer, 2011, 74(1): 7-11.
3.	Hirsch FR, Scagliotti GV, Mulshine JL, et al. Lung cancer: Current therapies and new targeted treatments. Lancet, 2017, 389(10066): 299-311.
4.	中华医学会放射肿瘤治疗学分会, 中国抗癌协会肿瘤放射治疗学专业委员会, 中国医师协会放射治疗医师分会. 早期非小细胞肺癌立体定向放疗中国专家共识(2019 版). 中华肿瘤杂志, 2020, 42(7): 522-530.
5.	Moumtzi D, Lampaki S, Zarogoulidis P, et al. Prognostic factors for long term survival in patients with advanced non-small cell lung cancer. Ann Transl Med, 2016, 4(9): 161.
6.	Devarakonda S, Morgensztern D, Govindan R. Genomic alterations in lung adenocarcinoma. Lancet Oncol, 2015, 16(7): e342-e351.
7.	Kim HP, Cho GA, Han SW, et al. Novel fusion transcripts in human gastric cancer revealed by transcriptome analysis. Oncogene, 2014, 33(47): 5434-5441.
8.	Nacu S, Yuan W, Kan Z, et al. Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples. BMC Med Genomics, 2011, 4: 11.
9.	Tang Y, Qin F, Liu A, et al. Recurrent fusion RNA DUS4L-BCAP29 in non-cancer human tissues and cells. Oncotarget, 2017, 8(19): 31415-31423.
10.	Tang Y, Guan F, Li H. Case study: The recurrent fusion RNA DUS4L-BCAP29 in noncancer human tissues and cells. Methods Mol Biol, 2020, 2079: 243-258.
11.	Tang Y, Ma S, Wang X, et al. Identification of chimeric RNAs in human infant brains and their implications in neural differentiation. Int J Biochem Cell Biol, 2019, 111: 19-26.
12.	Raine EV, Wreglesworth N, Dodd AW, et al. Gene expression analysis reveals HBP1 as a key target for the osteoarthritis susceptibility locus that maps to chromosome 7q22. Ann Rheum Dis, 2012, 71(12): 2020-2027.
13.	Hämäläinen S, Solovieva S, Vehmas T, et al. Genetic influences on hand osteoarthritis in Finnish women—A replication study of candidate genes. PLoS One, 2014, 9(5): e97417.
14.	Li Z, Yin C, Li B, et al. DUS4L silencing suppresses cell proliferation and promotes apoptosis in human lung adenocarcinoma cell line A549. Cancer Manag Res, 2020, 12: 9905-9913.
15.	Ieta K, Tanaka F, Yokobori T, et al. Clinicopathological significance of stanniocalcin 2 gene expression in colorectal cancer. Int J Cancer, 2009, 125(4): 926-931.
16.	Kita Y, Mimori K, Iwatsuki M, et al. STC2: A predictive marker for lymph node metastasis in esophageal squamous-cell carcinoma. Ann Surg Oncol, 2011, 18(1): 261-272.
17.	Meyer HA, Tölle A, Jung M, et al. Identification of stanniocalcin 2 as prognostic marker in renal cell carcinoma. Eur Urol, 2009, 55(3): 669-678.
18.	Yokobori T, Mimori K, Ishii H, et al. Clinical significance of stanniocalcin 2 as a prognostic marker in gastric cancer. Ann Surg Oncol, 2010, 17(10): 2601-2607.
19.	Na SS, Aldonza MB, Sung HJ, et al. Stanniocalcin-2 (STC2): A potential lung cancer biomarker promotes lung cancer metastasis and progression. Biochim Biophys Acta, 2015, 1854(6): 668-676.
20.	Zeiger W, Ito D, Swetlik C, et al. Stanniocalcin 2 is a negative modulator of store-operated calcium entry. Mol Cell Biol, 2011, 31(18): 3710-3722.
21.	Liu YN, Tsai MF, Wu SG, et al. Acquired resistance to EGFR tyrosine kinase inhibitors is mediated by the reactivation of STC2/JUN/AXL signaling in lung cancer. Int J Cancer, 2019, 145(6): 1609-1624.
22.	Yu JJ, Zhou DD, Yang XX, et al. TRIB3-EGFR interaction promotes lung cancer progression and defines a therapeutic target. Nat Commun, 2020, 11(1): 3660.
23.	Waugh DJ, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res, 2008, 14(21): 6735-6741.
24.	Koch AE, Polverini PJ, Kunkel SL, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science, 1992, 258(5089): 1798-1801.
25.	Li A, Dubey S, Varney ML, et al. IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol, 2003, 170(6): 3369-3376.
26.	Folkman J, Cotran R. Relation of vascular proliferation to tumor growth. Int Rev Exp Pathol, 1976, 16: 207-248.
27.	Yuan A, Yang PC, Yu CJ, et al. Interleukin-8 messenger ribonucleic acid expression correlates with tumor progression, tumor angiogenesis, patient survival, and timing of relapse in non-small-cell lung cancer. Am J Respir Crit Care Med, 2000, 162(5): 1957-1963.
28.	Smith DR, Polverini PJ, Kunkel SL, et al. Inhibition of interleukin 8 attenuates angiogenesis in bronchogenic carcinoma. J Exp Med, 1994, 179(5): 1409-1415.
29.	Arenberg DA, Kunkel SL, Polverini PJ, et al. Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice. J Clin Invest, 1996, 97(12): 2792-2802.
30.	Sanmamed MF, Carranza-Rua O, Alfaro C, et al. Serum interleukin-8 reflects tumor burden and treatment response across malignancies of multiple tissue origins. Clin Cancer Res, 2014, 20(22): 5697-5707.
31.	Fernando RI, Hamilton DH, Dominguez C, et al. IL-8 signaling is involved in resistance of lung carcinoma cells to erlotinib. Oncotarget, 2016, 7(27): 42031-42044.
32.	Dabkeviciene D, Jonusiene V, Zitkute V, et al. The role of interleukin-8 (CXCL8) and CXCR2 in acquired chemoresistance of human colorectal carcinoma cells HCT116. Med Oncol, 2015, 32(12): 258.
33.	Sanmamed MF, Perez-Gracia JL, Schalper KA, et al. Changes in serum interleukin-8 (IL-8) levels reflect and predict response to anti-PD-1 treatment in melanoma and non-small-cell lung cancer patients. Ann Oncol, 2017, 28(8): 1988-1995.
34.	Keskin S, Kutluk AC, Tas F. Prognostic and predictive role of angiogenic markers in non- small cell lung cancer. Asian Pac J Cancer Prev, 2019, 20(3): 733-736.
35.	Fousek K, Horn LA, Palena C. Interleukin-8: A chemokine at the intersection of cancer plasticity, angiogenesis, and immune suppression. Pharmacol Ther, 2021, 219: 107692.
36.	Horn LA, Riskin J, Hempel HA, et al. Simultaneous inhibition of CXCR1/2, TGF-β, and PD-L1 remodels the tumor and its microenvironment to drive antitumor immunity. J Immunother Cancer, 2020, 8(1): e000326.
37.	Sun L, Clavijo PE, Robbins Y, et al. Inhibiting myeloid-derived suppressor cell trafficking enhances T cell immunotherapy. JCI Insight, 2019, 4(7): e126853.

1. Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2021, 71(3): 209-249.
2. Yatabe Y, Borczuk AC, Powell CA. Do all lung adenocarcinomas follow a stepwise progression? Lung Cancer, 2011, 74(1): 7-11.
3. Hirsch FR, Scagliotti GV, Mulshine JL, et al. Lung cancer: Current therapies and new targeted treatments. Lancet, 2017, 389(10066): 299-311.
4. 中华医学会放射肿瘤治疗学分会, 中国抗癌协会肿瘤放射治疗学专业委员会, 中国医师协会放射治疗医师分会. 早期非小细胞肺癌立体定向放疗中国专家共识(2019 版). 中华肿瘤杂志, 2020, 42(7): 522-530.
5. Moumtzi D, Lampaki S, Zarogoulidis P, et al. Prognostic factors for long term survival in patients with advanced non-small cell lung cancer. Ann Transl Med, 2016, 4(9): 161.
6. Devarakonda S, Morgensztern D, Govindan R. Genomic alterations in lung adenocarcinoma. Lancet Oncol, 2015, 16(7): e342-e351.
7. Kim HP, Cho GA, Han SW, et al. Novel fusion transcripts in human gastric cancer revealed by transcriptome analysis. Oncogene, 2014, 33(47): 5434-5441.
8. Nacu S, Yuan W, Kan Z, et al. Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples. BMC Med Genomics, 2011, 4: 11.
9. Tang Y, Qin F, Liu A, et al. Recurrent fusion RNA DUS4L-BCAP29 in non-cancer human tissues and cells. Oncotarget, 2017, 8(19): 31415-31423.
10. Tang Y, Guan F, Li H. Case study: The recurrent fusion RNA DUS4L-BCAP29 in noncancer human tissues and cells. Methods Mol Biol, 2020, 2079: 243-258.
11. Tang Y, Ma S, Wang X, et al. Identification of chimeric RNAs in human infant brains and their implications in neural differentiation. Int J Biochem Cell Biol, 2019, 111: 19-26.
12. Raine EV, Wreglesworth N, Dodd AW, et al. Gene expression analysis reveals HBP1 as a key target for the osteoarthritis susceptibility locus that maps to chromosome 7q22. Ann Rheum Dis, 2012, 71(12): 2020-2027.
13. Hämäläinen S, Solovieva S, Vehmas T, et al. Genetic influences on hand osteoarthritis in Finnish women—A replication study of candidate genes. PLoS One, 2014, 9(5): e97417.
14. Li Z, Yin C, Li B, et al. DUS4L silencing suppresses cell proliferation and promotes apoptosis in human lung adenocarcinoma cell line A549. Cancer Manag Res, 2020, 12: 9905-9913.
15. Ieta K, Tanaka F, Yokobori T, et al. Clinicopathological significance of stanniocalcin 2 gene expression in colorectal cancer. Int J Cancer, 2009, 125(4): 926-931.
16. Kita Y, Mimori K, Iwatsuki M, et al. STC2: A predictive marker for lymph node metastasis in esophageal squamous-cell carcinoma. Ann Surg Oncol, 2011, 18(1): 261-272.
17. Meyer HA, Tölle A, Jung M, et al. Identification of stanniocalcin 2 as prognostic marker in renal cell carcinoma. Eur Urol, 2009, 55(3): 669-678.
18. Yokobori T, Mimori K, Ishii H, et al. Clinical significance of stanniocalcin 2 as a prognostic marker in gastric cancer. Ann Surg Oncol, 2010, 17(10): 2601-2607.
19. Na SS, Aldonza MB, Sung HJ, et al. Stanniocalcin-2 (STC2): A potential lung cancer biomarker promotes lung cancer metastasis and progression. Biochim Biophys Acta, 2015, 1854(6): 668-676.
20. Zeiger W, Ito D, Swetlik C, et al. Stanniocalcin 2 is a negative modulator of store-operated calcium entry. Mol Cell Biol, 2011, 31(18): 3710-3722.
21. Liu YN, Tsai MF, Wu SG, et al. Acquired resistance to EGFR tyrosine kinase inhibitors is mediated by the reactivation of STC2/JUN/AXL signaling in lung cancer. Int J Cancer, 2019, 145(6): 1609-1624.
22. Yu JJ, Zhou DD, Yang XX, et al. TRIB3-EGFR interaction promotes lung cancer progression and defines a therapeutic target. Nat Commun, 2020, 11(1): 3660.
23. Waugh DJ, Wilson C. The interleukin-8 pathway in cancer. Clin Cancer Res, 2008, 14(21): 6735-6741.
24. Koch AE, Polverini PJ, Kunkel SL, et al. Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science, 1992, 258(5089): 1798-1801.
25. Li A, Dubey S, Varney ML, et al. IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis. J Immunol, 2003, 170(6): 3369-3376.
26. Folkman J, Cotran R. Relation of vascular proliferation to tumor growth. Int Rev Exp Pathol, 1976, 16: 207-248.
27. Yuan A, Yang PC, Yu CJ, et al. Interleukin-8 messenger ribonucleic acid expression correlates with tumor progression, tumor angiogenesis, patient survival, and timing of relapse in non-small-cell lung cancer. Am J Respir Crit Care Med, 2000, 162(5): 1957-1963.
28. Smith DR, Polverini PJ, Kunkel SL, et al. Inhibition of interleukin 8 attenuates angiogenesis in bronchogenic carcinoma. J Exp Med, 1994, 179(5): 1409-1415.
29. Arenberg DA, Kunkel SL, Polverini PJ, et al. Inhibition of interleukin-8 reduces tumorigenesis of human non-small cell lung cancer in SCID mice. J Clin Invest, 1996, 97(12): 2792-2802.
30. Sanmamed MF, Carranza-Rua O, Alfaro C, et al. Serum interleukin-8 reflects tumor burden and treatment response across malignancies of multiple tissue origins. Clin Cancer Res, 2014, 20(22): 5697-5707.
31. Fernando RI, Hamilton DH, Dominguez C, et al. IL-8 signaling is involved in resistance of lung carcinoma cells to erlotinib. Oncotarget, 2016, 7(27): 42031-42044.
32. Dabkeviciene D, Jonusiene V, Zitkute V, et al. The role of interleukin-8 (CXCL8) and CXCR2 in acquired chemoresistance of human colorectal carcinoma cells HCT116. Med Oncol, 2015, 32(12): 258.
33. Sanmamed MF, Perez-Gracia JL, Schalper KA, et al. Changes in serum interleukin-8 (IL-8) levels reflect and predict response to anti-PD-1 treatment in melanoma and non-small-cell lung cancer patients. Ann Oncol, 2017, 28(8): 1988-1995.
34. Keskin S, Kutluk AC, Tas F. Prognostic and predictive role of angiogenic markers in non- small cell lung cancer. Asian Pac J Cancer Prev, 2019, 20(3): 733-736.
35. Fousek K, Horn LA, Palena C. Interleukin-8: A chemokine at the intersection of cancer plasticity, angiogenesis, and immune suppression. Pharmacol Ther, 2021, 219: 107692.
36. Horn LA, Riskin J, Hempel HA, et al. Simultaneous inhibition of CXCR1/2, TGF-β, and PD-L1 remodels the tumor and its microenvironment to drive antitumor immunity. J Immunother Cancer, 2020, 8(1): e000326.
37. Sun L, Clavijo PE, Robbins Y, et al. Inhibiting myeloid-derived suppressor cell trafficking enhances T cell immunotherapy. JCI Insight, 2019, 4(7): e126853.

Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

Effect of DUS4L knockdown on gene expression regulation of human A549 lung adenocarcinoma cell line and analysis of different genes

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