Establishment of a Livin-gene silencing system and its effect on the sensitivity of Cal 27 with regard to 5-fluorouracil

Volume 6, Issue 6, December 2021     |     PP. 474-487      |     PDF (481 K)    |     Pub. Date: October 19, 2021
DOI: 10.54647/cm32648    81 Downloads     3312 Views  


Zixiao Huang, School of Dentistry, Lanzhou University, Lanzhou, 730000, P. R. China;Xiangyang Stomatological Hospital; Affiliated Stomatological Hospital of Hubei University of Arts and Science, Xiangyang, 441000, P. R. China
Ziwei Cui, School of Dentistry, Lanzhou University, Lanzhou, 730000, P. R. China
Ruoshan Qin, School of Dentistry, Lanzhou University, Lanzhou, 730000, P. R. China
Xuanxuan Yao, School of Dentistry, Lanzhou University, Lanzhou, 730000, P. R. China
Hongli Zhou, School of Dentistry, Lanzhou University, Lanzhou, 730000, P. R. China
Ru Guo, School of Dentistry, Lanzhou University, Lanzhou, 730000, P. R. China
Xiaodong Qin, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Sciences, Lanzhou, Gansu, 730000, P. R. China
Xiangyi He, School of Dentistry, Lanzhou University, Lanzhou, 730000, P. R. China;Key Laboratory of Functional Genomic and Molecular Diagnosis of Gansu Province, Lanzhou, 730030, P. R. China

Objective: This study aims at downregulating the expression of Livin through shRNA and exploring the sensitivity of Cal 27 to 5-fluorouracil (5-FU), so as to provide some help in resolving the chemotherapy resistance of oral cancer.
Methods: The Livin-targeting shRNA sequence was designed to construct a lentivirus and infect Cal 27 to obtain a stable Livin-silencing cell strain. After being treated with 20 μM, 40 μM and 60 μM of 5-fluorouracil for 24 h, cell apoptosis and cell viability were measured by flow cytometry and MTT assay, respectively.
Results: A shRNA-based lentivirus targeting Livin was successfully designed and constructed. After the silencing of Livin, the sensitivity and cell apoptosis of Cal 27 to 5-FU were dramatically elevated, and cell viability was significantly reduced (P<0.05). Moreover, the inhibition of Caspase 3 was observed.
Conclusion: The expression level of Livin was downregulated in Cal 27 after shRNA transfection, which increased the sensitivity of Cal to 5-FU. The underlying mechanism is the blockage of the activation of Caspase 3. Therefore, Livin may serve as a promising target for oral cancer treatment.

Mouth Neoplasms; Inhibitor of Apoptosis Proteins; Drug Therapy; Caspase 3; Fluorouracil; Gene Silencing

Cite this paper
Zixiao Huang, Ziwei Cui, Ruoshan Qin, Xuanxuan Yao, Hongli Zhou, Ru Guo, Xiaodong Qin, Xiangyi He, Establishment of a Livin-gene silencing system and its effect on the sensitivity of Cal 27 with regard to 5-fluorouracil , SCIREA Journal of Clinical Medicine. Volume 6, Issue 6, December 2021 | PP. 474-487. 10.54647/cm32648


[ 1 ] Bray, F. et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians 68, 394-424, doi:10.3322/caac.21492 (2018).
[ 2 ] Chen, W. et al. Cancer statistics in China, 2015. CA: a cancer journal for clinicians 66, 115-132, doi:10.3322/caac.21338 (2016).
[ 3 ] Ferlay, J. et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 136, E359-386, doi:10.1002/ijc.29210 (2015).
[ 4 ] Shield, K. D. et al. The global incidence of lip, oral cavity, and pharyngeal cancers by subsite in 2012. CA: a cancer journal for clinicians 67, 51-64, doi:10.3322/caac.21384 (2017).
[ 5 ] Azharuddin, M. et al. Dissecting multi drug resistance in head and neck cancer cells using multicellular tumor spheroids. Scientific Reports 9, 20066, doi:10.1038/s41598-019-56273-6 (2019).
[ 6 ] Vasan, N., Baselga, J. & Hyman, D. M. A view on drug resistance in cancer. Nature 575, 299-309, doi:10.1038/s41586-019-1730-1 (2019).
[ 7 ] Bell, C. C. & Gilan, O. Principles and mechanisms of non-genetic resistance in cancer. British Journal of Cancer, doi:10.1038/s41416-019-0648-6 (2019).
[ 8 ] Altieri, B. et al. Livin/BIRC7 expression as malignancy marker in adrenocortical tumors. Oncotarget 8, 9323-9338, doi:10.18632/oncotarget.14067 (2017).
[ 9 ] Wang, H. et al. Single-chain antibody-delivered Livin siRNA inhibits human malignant melanoma growth in vitro and in vivo. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 39, 1010428317701645, doi:10.1177/1010428317701645 (2017).
[ 10 ] Su, Q. B. et al. Livin serves as a prognostic marker for mid-distal rectal cancer and a target of mid-distal rectal cancer treatment. Oncology letters 14, 7759-7766, doi:10.3892/ol.2017.7230 (2017).
[ 11 ] Wang, Z. et al. Silencing Livin induces apoptotic and autophagic cell death, increasing chemotherapeutic sensitivity to cisplatin of renal carcinoma cells. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 37, 15133-15143, doi:10.1007/s13277-016-5395-1 (2016).
[ 12 ] Zhuang, L. et al. Inhibition of livin expression suppresses cell proliferation and enhances chemosensitivity to cisplatin in human lung adenocarcinoma cells. Molecular medicine reports 12, 547-552, doi:10.3892/mmr.2015.3372 (2015).
[ 13 ] Elmekkawy, B. K., Shoaib, R. M. S., Seleem, A. K., Shalaan, D. & Saad, E. A. Livin/BIRC7 gene expression as a possible diagnostic biomarker for endometrial hyperplasia and carcinoma. Journal, genetic engineering & biotechnology 19, 141, doi:10.1186/s43141-021-00244-w (2021).
[ 14 ] Fung, S., Knoefel, W. T. & Krieg, A. Clinicopathological and Prognostic Significance of Inhibitor of Apoptosis Protein (IAP) Family Members in Lung Cancer: A Meta-Analysis. Cancers 13, doi:10.3390/cancers13164098 (2021).
[ 15 ] Ge, Y., Liu, B.-l., Cui, J.-p. & Li, S.-q. Livin promotes colon cancer progression by regulation of H2A.XY39ph via JMJD6. Life Sciences 234, 116788, doi: (2019).
[ 16 ] Liu, S. et al. Silencing Livin improved the sensitivity of colon cancer cells to 5-fluorouracil by regulating crosstalk between apoptosis and autophagy. Oncology letters 15, 7707-7715, doi:10.3892/ol.2018.8282 (2018).
[ 17 ] Uegaki, T. et al. Inhibitor of apoptosis proteins (IAPs) may be effective therapeutic targets for treating endometriosis. Human reproduction (Oxford, England) 30, 149-158, doi:10.1093/humrep/deu288 (2015).
[ 18 ] Xue, J. M. et al. Livin in synergy with Ras induces and sustains corticosteroid resistance in the airway mucosa. International journal of biological sciences 17, 2089-2098, doi:10.7150/ijbs.58427 (2021).
[ 19 ] Jin, J. et al. Bioinformatics analysis of aberrantly expressed exosomal lncRNAs in oral squamous cell carcinoma (CAL-27 vs. oral epithelial) cells. Oncology letters 20, 2378-2386, doi:10.3892/ol.2020.11764 (2020).
[ 20 ] Qin, X. et al. Mechanism and significance of apoptosis of the immortalized human oral mucosal epithelial cells established by Lentivirus-mediated hTERT. Mol Biol Rep 47, 5469-5475, doi:10.1007/s11033-020-05637-7 (2020).
[ 21 ] Sun, G. et al. Comparison of Periodontal Ligament Cell Lines with Adenovirus- and Lentivirus-Mediated Human Telomerase Reverse Transcription Expression. Human gene therapy methods 30, 53-59, doi:10.1089/hgtb.2018.184 (2019).
[ 22 ] Hrdinka, M. & Yabal, M. Inhibitor of apoptosis proteins in human health and disease. Genes and immunity 20, 641-650, doi:10.1038/s41435-019-0078-8 (2019).
[ 23 ] Nakajima, Y. I. & Kuranaga, E. Caspase-dependent non-apoptotic processes in development. Cell death and differentiation 24, 1422-1430, doi:10.1038/cdd.2017.36 (2017).
[ 24 ] Li, P. et al. Caspase-9: structure, mechanisms and clinical application. Oncotarget 8, 23996-24008, doi:10.18632/oncotarget.15098 (2017).
[ 25 ] Green, D. R. & Llambi, F. Cell Death Signaling. Cold Spring Harbor perspectives in biology 7, doi:10.1101/cshperspect.a006080 (2015).
[ 26 ] Kasof, G. M. & Gomes, B. C. Livin, a novel inhibitor of apoptosis protein family member. J Biol Chem 276, 3238-3246, doi:10.1074/jbc.M003670200 (2001).
[ 27 ] Jin, F. et al. Difference in the Inhibitory Effect of Temozolomide on TJ905 Glioma Cells and Stem Cells. Frontiers in neurology 8, 474, doi:10.3389/fneur.2017.00474 (2017).
[ 28 ] Yang, D. et al. Therapeutic potential of siRNA-mediated combined knockdown of the IAP genes (Livin, XIAP, and Survivin) on human bladder cancer T24 cells. Acta biochimica et biophysica Sinica 42, 137-144, doi:10.1093/abbs/gmp118 (2010).
[ 29 ] Li, G. et al. Effect of temozolomide on livin and caspase-3 in U251 glioma stem cells. Experimental and therapeutic medicine 9, 744-750, doi:10.3892/etm.2014.2144 (2015).
[ 30 ] Liang, Y. et al. miR-198-induced upregulation of Livin may be associated with the prognosis and contribute to the oncogenesis of lung adenocarcinoma. Oncol Rep 38, 2096-2104, doi:10.3892/or.2017.5866 (2017).