Knockdown of RFC4 inhibits the cell proliferation of nasopharyngeal carcinoma in vitro and in vivo

Abstract

Nasopharyngeal carcinoma (NPC) is a malignant tumor that mainly occurs in East and Southeast Asia. Although patients benefit from the main NPC treatments (e.g., radiotherapy and concurrent chemotherapy), persistent and recurrent diseases still occur in some NPC patients. Therefore, investigating the pathogenesis of NPC is of great clinical significance. In the present study, replication factor c subunit 4 (RFC4) is a key potential target involved in NPC progression via bioinformatics analysis. Furthermore, the expression and mechanism of RFC4 in NPC were investigated in vitro and in vivo. Our results revealed that RFC4 was more elevated in NPC tumor tissues than in normal tissues. RFC4 knockdown induced G2/M cell cycle arrest and inhibited NPC cell proliferation in vitro and in vivo. Interestingly, HOXA10 was confirmed as a downstream target of RFC4, and the overexpression of HOXA10 attenuated the silencing of RFC4-induced cell proliferation, colony formation inhibition, and cell cycle arrest. For the first time, this study reveals that RFC4 is required for NPC cell proliferation and may play a pivotal role in NPC tumorigenesis.

Keywords

nasopharyngeal carcinoma / WGCNA / RFC4 / proliferation

References

[1] Chen YP, Chan ATC, Le QT, Blanchard P, Sun Y, Ma J. Nasopharyngeal carcinoma. Lancet 2019; 394(10192): 64–80

[2] Wang M, Gu B, Chen X, Wang Y, Li P, Wang K. The function and therapeutic potential of Epstein-Barr virus-encoded microRNAs in cancer. Mol Ther Nucleic Acids 2019; 17: 657–668

[3] Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. 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

[4] Chen YP, Ismaila N, Chua MLK, Colevas AD, Haddad R, Huang SH, Wee JTS, Whitley AC, Yi JL, Yom SS, Chan ATC, Hu CS, Lang JY, Le QT, Lee AWM, Lee N, Lin JC, Ma B, Morgan TJ, Shah J, Sun Y, Ma J. Chemotherapy in combination with radiotherapy for definitive-intent treatment of stage II-IVA nasopharyngeal carcinoma: CSCO and ASCO Guideline. J Clin Oncol 2021; 39(7): 840–859

[5] Guan S, Wei J, Huang L, Wu L. Chemotherapy and chemo-resistance in nasopharyngeal carcinoma. Eur J Med Chem 2020; 207: 112758

[6] Wang S, Claret FX, Wu W. MicroRNAs as therapeutic targets in nasopharyngeal carcinoma. Front Oncol 2019; 9: 756

[7] Bowman GD, O’Donnell M, Kuriyan J. Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex. Nature 2004; 429(6993): 724–730

[8] Okumura K, Nogami M, Taguchi H, Dean FB, Chen M, Pan ZQ, Hurwitz J, Shiratori A, Murakami Y, Ozawa K, Eki T. Assignment of the 36.5-kDa (RFC5), 37-kDa (RFC4), 38-kDa (RFC3), and 40-kDa (RFC2) subunit genes of human replication factor C to chromosome bands 12q24.2-q24.3, 3q27, 13q12.3-q13, and 7q11.23. Genomics 1995; 25(1): 274–278

[9] Gaubitz C, Liu X, Magrino J, Stone NP, Landeck J, Hedglin M, Kelch BA. Structure of the human clamp loader reveals an autoinhibited conformation of a substrate-bound AAA+ switch. Proc Natl Acad Sci USA 2020; 117(38): 23571–23580

[10] Zhu W, Zhang Q, Liu M, Yan M, Chu X, Li Y. Identification of DNA repair-related genes predicting pathogenesis and prognosis for liver cancer. Cancer Cell Int 2021; 21(1): 81

[11] Xiang J, Fang L, Luo Y, Yang Z, Liao Y, Cui J, Huang M, Yang Z, Huang Y, Fan X, Wang H, Wang L, Peng J, Wang J. Levels of human replication factor C4, a clamp loader, correlate with tumor progression and predict the prognosis for colorectal cancer. J Transl Med 2014; 12(1): 320

[12] Zhang J, Wang L, Xie X. RFC4 promotes the progression and growth of oral tongue squamous cell carcinoma in vivo and vitro. J Clin Lab Anal 2021; 35(5): e23761

[13] Arai M, Kondoh N, Imazeki N, Hada A, Hatsuse K, Matsubara O, Yamamoto M. The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cells. Liver Int 2009; 29(1): 55–62

[14] Wang XC, Yue X, Zhang RX, Liu TY, Pan ZZ, Yang MJ, Lu ZH, Wang ZY, Peng JH, Le LY, Wang GY, Peng QH, Meng Y, Huang W, Liu RY. Genome-wide RNAi screening identifies RFC4 as a factor that mediates radioresistance in colorectal cancer by facilitating nonhomologous end joining repair. Clin Cancer Res 2019; 25(14): 4567–4579

[15] Bushweller JH. Targeting transcription factors in cancer—from undruggable to reality. Nat Rev Cancer 2019; 19(11): 611–624

[16] Xie L, Xu L, He Z, Zhou W, Wang L, Zhang L, Lan K, Ren C, Liu W, Yao K. Identification of differentially expressed genes in nasopharyngeal carcinoma by means of the Atlas human cancer cDNA expression array. J Cancer Res Clin Oncol 2000; 126(7): 400–406

[17] Zhu Q, Zhang Q, Gu M, Zhang K, Xia T, Zhang S, Chen W, Yin H, Yao H, Fan Y, Pan S, Xie H, Liu H, Cheng T, Zhang P, Zhang T, You B, You Y. MIR106A-5p upregulation suppresses autophagy and accelerates malignant phenotype in nasopharyngeal carcinoma. Autophagy 2021; 17(7): 1667–1683

[18] Sang Y, Cheng C, Zeng YX, Kang T. Snail promotes metastasis of nasopharyngeal carcinoma partly by down-regulating TEL2. Cancer Commun (Lond) 2018; 38(1): 58

[19] Shikata M, Ishikawa F, Kanoh J. Tel2 is required for activation of the Mrc1-mediated replication checkpoint. J Biol Chem 2007; 282(8): 5346–5355

[20] Sang Y, Chen MY, Luo D, Zhang RH, Wang L, Li M, Luo R, Qian CN, Shao JY, Zeng YX, Kang T. TEL2 suppresses metastasis by down-regulating SERPINE1 in nasopharyngeal carcinoma. Oncotarget 2015; 6(30): 29240–29253

[21] Li H, You J, Xue H, Tan X, Chao C. CircCTDP1 promotes nasopharyngeal carcinoma progression via a microRNA-320b/HOXA10/TGFβ2 pathway. Int J Mol Med 2020; 45(3): 836–846

[22] Shen ZH, Zhao KM, Du T. HOXA10 promotes nasopharyngeal carcinoma cell proliferation and invasion via inducing the expression of ZIC2. Eur Rev Med Pharmacol Sci 2017; 21(5): 945–952

[23] Huang W, Song W, Jiang Y, Chen L, Lu H. c-Myc-induced circ-NOTCH1 promotes aggressive phenotypes of nasopharyngeal carcinoma cells by regulating the miR-34c-5p/c-Myc axis. Cell Biol Int 2021; 45(7): 1436–1447

[24] Liu J, Wen Y, Liu Z, Liu S, Xu P, Xu Y, Deng S, Hu S, Luo R, Jiang J, Yu G. VPS33B modulates c-Myc/p53/miR-192-3p to target CCNB1 suppressing the growth of non-small cell lung cancer. Mol Ther Nucleic Acids 2021; 23: 324–335

[25] Chung GT, Lou WP, Chow C, To KF, Choy KW, Leung AW, Tong CY, Yuen JW, Ko CW, Yip TT, Busson P, Lo KW. Constitutive activation of distinct NF-κB signals in EBV-associated nasopharyngeal carcinoma. J Pathol 2013; 231(3): 311–322

[26] Ma F, Zhi C, Wang M, Li T, Khan SA, Ma Z, Jing Z, Bo C, Zhou Q, Xia S, Huang S, Huang S, Zhang Z, Jia H, Cui X, Yao M, Ji T. Dysregulated NF-κB signal promotes the hub gene PCLAF expression to facilitate nasopharyngeal carcinoma proliferation and metastasis. Biomed Pharmacother 2020; 125: 109905

[27] Huang YX, Chen XT, Guo KY, Li YH, Wu BY, Song CY, He YJ. Sunitinib induces NK-κB-dependent NKG2D ligand expression in nasopharyngeal carcinoma and hepatoma cells. J Immunother 2017; 40(5): 164–174

[28] Ren ZH, Shang GP, Wu K, Hu CY, Ji T. WGCNA co-expression network analysis reveals ILF3-AS1 functions as a ceRNA to regulate PTBP1 expression by sponging miR-29a in gastric cancer. Front Genet 2020; 11: e.g.39

[29] Tian F, Zhao J, Fan X, Kang Z. Weighted gene co-expression network analysis in identification of metastasis-related genes of lung squamous cell carcinoma based on the Cancer Genome Atlas database. J Thorac Dis 2017; 9(1): 42–53

[30] Liu J, Li S, Lin L, Jiang Y, Wan Y, Zhou S, Cheng W. Co-expression network analysis identified atypical chemokine receptor 1 (ACKR1) association with lymph node metastasis and prognosis in cervical cancer. Cancer Biomark 2020; 27(2): 213–223

[31] Cui H, Wang Q, Lei Z, Feng M, Zhao Z, Wang Y, Wei G. DTL promotes cancer progression by PDCD4 ubiquitin-dependent degradation. J Exp Clin Cancer Res 2019; 38(1): 350

[32] Luo Y, Qu X, Kan D, Cai B. The microRNA-451a/chromosome segregation 1-like axis suppresses cell proliferation, migration, and invasion and induces apoptosis in nasopharyngeal carcinoma. Bioengineered 2021; 12(1): 6967–6980

[33] Wang B, Wang W, Wang H, Liu W. Microarray analysis of novel genes involved in nasopharyngeal carcinoma. Bull Exp Biol Med 2021; 170(5): 658–664

[34] Ding X, Duan H, Luo H. Identification of core gene expression signature and key pathways in colorectal cancer. Front Genet 2020; 11: 45

[35] Chen W, Zhu S, Zhang Y, Xiao J, Tian D. Identification of key candidate tumor biomarkers in non-small-cell lung cancer by in silico analysis. Oncol Lett 2020; 19(1): 1008–1016

[36] Qiu HZ, Huang J, Xiang CC, Li R, Zuo ED, Zhang Y, Shan L, Cheng X. Screening and discovery of new potential biomarkers and small molecule drugs for cervical cancer: a bioinformatics analysis. Technol Cancer Res Treat 2020; 19: 1533033820980112

[37] Yoshida H, Broaddus R, Cheng W, Xie S, Naora H. Deregulation of the HOXA10 homeobox gene in endometrial carcinoma: role in epithelial-mesenchymal transition. Cancer Res 2006; 66(2): 889–897

[38] Hatanaka Y, de Velasco MA, Oki T, Shimizu N, Nozawa M, Yoshimura K, Yoshikawa K, Nishio K, Uemura H. HOXA10 expression profiling in prostate cancer. Prostate 2019; 79(5): 554–563

[39] Han Y, Lu S, Wen YG, Yu FD, Zhu XW, Qiu GQ, Tang HM, Peng ZH, Zhou CZ. Overexpression of HOXA10 promotes gastric cancer cells proliferation and HOXA10⁺/CD44⁺ is potential prognostic biomarker for gastric cancer. Eur J Cell Biol 2015; 94(12): 642–652

[40] Gong D, Zhu H, Zeng L, Hu R, Hu J, Ding J. Overexpression of HOXA10 promotes the growth and metastasis of nasopharyngeal carcinoma. Exp Biol Med (Maywood) 2021; 246(23): 2454–2462

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