OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Downregulation of NSD3 (WHSC1L1) inhibits cell proliferation and migration via ERK1/2 deactivation and decreasing CAPG expression in colorectal cancer cells This article was published in the following Dove Press journal: OncoTargets and Therapy Lanjuan Yi1 Purpose: NSD3 (WHSC1L1) is a protein lysine methyltransferase that is recurrently Lanjie Yi2 amplified (8p11.23) in several cancer types, and its upregulation is involved in tumor cell Qing Liu3 proliferation, metastasis, and epithelial-mesenchymal transition (EMT). We aimed to evalu- Chen Li4 ate its potential function as an oncogenic force in colorectal cancer (CRC), and to elucidate relevant mechanisms of its oncogenic activity. 1Department of gastroenterology, Yantai Shan Hospital, Yantai, Shandong 264001, Materials and methods: NSD3 levels were analyzed in human CRC and adjacent normal People’s Republic of China; 2Research tissues or cells by Western blot analysis and RT-qPCR. Expression levels of the proteins were Office of Clinical literature, Nanjing detected by Western blot analysis and RT-qPCR. University of Chinese Medicine, Nanjing, fi Jiangsu 210023, People’s Republic of Results: NSD3 was signi cantly upregulated in both CRC tissues and cell lines. China; 3Department of Nosocomial Knockdown of NSD3 expression resulted in significant decreases in CRC cell proliferation, Infection Control, Xuzhou Hospital of migration, and EMT process marker proteins vimentin, simultaneously reducing E-cadherin Traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, and N-cadherin expression. The opposite results were observed when NSD3 was over- Xuzhou, Jiangsu 310015, People’s expressed. Additionally, overexpressing of NSD3 dramatically activated the extracellular Republic of China; 4Department of signal-regulated kinase 1/2 (ERK1/2) signaling pathway and enhanced actin-capping protein Gastroenterology, Xuzhou Chinese Medicine Hospital Affiliated to Nanjing (CAPG) expression. Furthermore, the proliferation and migration abilities evidently facili- University of Chinese Medicine, Xuzhou, tated by pcDNA3.1(+) expression vector containing full-length CDS of NSD3 (pcDNA3.1 Jiangsu 310015, People’s Republic of China (+)-NSD3, or NSD3) were partially decreased after incubation with ERK1/2 signaling path- way inhibitor (PD98059) and/or specific siRNA against CAPG (siCAPG) in SW480 and HT- 29 CRC cells. Conclusion: NSD3 overexpression stimulated CRC cell proliferation and migration through targeting the ERK1/2 signaling pathway and downstream CAPG. Thus, NSD3 could serve as a promising target for anticancer drug development for patients with CRC. Keywords: colorectal cancer, histone methyltransferase NSD3, extracellular signal- regulated kinase 1/2, actin-capping protein (CAPG) Plain Language Summary Correspondence: Chen Li The purpose of the experiment is to evaluate potential function of NSD3 Department of Gastroenterology, Xuzhou Chinese Medicine Hospital (WHSC1L1), aprotein lysine methyltransferase, as an oncogenic force in colorectal Affiliated to Nanjing University of Chinese cancer (CRC), and to elucidate relevant mechanisms of its oncogenic activity. Our Medicine, No.169, Zhongshan South Road, Xuzhou, Jiangsu 221000, People’s invitro studies revealed that the expression of NSD3 was obviously higher in CRC Republic of China tissues than in normal tissues. Knockdown of NSD3 expression resulted in sig- Tel+ 86 0516 6869 2031 Email [email protected] nificant decreases in CRC cell proliferation, migration, and epithelial-mesenchymal submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 3933–3943 3933 DovePress © 2019 Yi et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work http://doi.org/10.2147/OTT.S191732 you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Yi et al Dovepress transition (EMT) process marker proteins vimentin, simul- we examined its effect on the regulation of extracellular taneously reducing E-cadherin and N-cadherin expression. signal regulated kinases 1 and 2 (ERK1/2) signaling path- The opposite results were observed when NSD3 was over- way and actin-capping protein (CAPG), which are strongly expressed. NSD3 overexpression in CRC cells facilitated associated with tumor cell proliferation, apoptosis, metas- cell proliferation and migration, and promoted cell EMT tasis, and EMT.15–18 progress via increasing CAPG expression and ERK1/2 signaling pathway activation. Additionally, we also Materials and methods demonstrated that ERK1/2 signaling pathway could facil- Human CRC tissues and cell lines itate CAPG-induced proliferation and migration in CRC In total, CRC tissues (n =24) and adjacent normal tissues cells. These results imply NSD3 may become a reliable (n =26) were collected after surgical resection at the CRC biomarker for diagnoses and atarget for precise Xuzhou Hospital of Traditional Chinese Medicine therapy. Affiliated to Nanjing University of Chinese Medicine Introduction (Xuzhou, Jiangsu, China).All samples were immediately snap-frozen in liquid nitrogen and stored at −80 °C until Colorectal cancer (CRC) is a type of gastrointestinal neo- total RNA was extracted. The researches were approved by plasm with the highest rates both in incidence and the Independent Ethics Committee (IEC) of Xuzhou mortality.1 More than 49 million patients worldwide die Hospital of Traditional Chinese Medicine Affiliated to of CRC each year.2,3 In China, the morbidity of CRC has Nanjing University of Chinese Medicine, and all patients been increasing since 2000.4 As most CRC cases are provided written informed consent. The study methodolo- diagnosed at metastatic stages, the prognosis of CRC gies conformed to the standards set by the Declaration of patients is poor.5 Therefore, it is urgent to develop early Helsinki. diagnose and effective intervene preventing tumors from The human normal colorectal mucosa cell line (FHC) metastasize in CRC. and seven human CRC cell lines, including Lovo, SW480, It has been widely identified that aberrant expression of SW620, HT-29, HCT-116, Caco-2, and SW48, were all histone methylation and its regulator histone methyltrans- purchased from the Cell Bank of the Chinese Academy of ferase are closely related to various cancers.6 NSD3, also Sciences (Shanghai, China). Cells were cultured in high- known as Wolf - Hirschhorn syndrome candidate 1-like 1 glucose Dulbecco’s modified Eagle’s medium (Invitrogen, (WHSC1L1), is a nuclear protein mapped at chromosome Carlsbad, CA, USA) with 10% fetal bovine serum 8p11.23 and functions as a chromatin regulator by mod- (Hyclone Laboratories, Inc., South Logan, UT, USA) and ulating the expression of genes through demethylation of 100 μg/mL streptomycin sulfate (Gibco, Rockville, MD, lysine 36 on histone H3 (H3K36me2).7 NSD3 plays USA) in plastic tissue culture plates. All cells were main- a critical role in the expansion of various tumors.8 It can tained at 37 °C in a humidified atmosphere containing 5% interact with NUP98,9 NUT,10 E2F2,11 BRD4, or CHD8,12 CO . resulting in occurrence of tumors or proliferation and 2 invasion of cancer cells in leukemogenesis, NUT midline carcinoma (NMC), and breast cancer. Collectively, NSD3 RNA extraction and quantitative real-time plays an important role in human carcinogenesis, and it reverse transcription PCR (RT-qPCR) may serve as a potential druggable target for selective Total RNA was extracted using TRIzol Reagent (Invitrogen, CRC therapy in the future. However, the functional Carlsbad, CA, USA), and cDNA was synthesized from 2 μg mechanisms of NSD3 in CRC have not been clarified yet. of RNA using the PrimeScriptTM RT Reagent Kit (Takara Current findings show that epithelial-mesenchymal Biotechnology, Dalian, China) according to the manufac- transition (EMT) is an important mechanism for the metas- turer’s instructions. The sequences for sense (S) and tasis of malignant tumors.13 By EMT epithelial-derived antisense (AS) primers are as follows: human-NSD3-S, malignant cells acquire the phenotype of mesenchymal 5′-AAG AGC CAC CGC CTG TTA AA-3′, human-NSD3- cell and the ability of invasion and metastasis.14 Herein, AS, 5′- GCT GTC ACA AAT GGA GGT GC-3′; human- we investigated the effects of NSD3 on CRC cell prolif- E-cadherin-S, 5′-CGG GAA TGC AGT TGA GGA TC-3′, eration, metastasis and EMT progress, and then explored human-E-cadherin-AS, 5′-AGG ATG GTG TAA GCG ATG the molecular mechanism of NSD3 in CRC. Specifically, GC-3′; human-N-cadherin-S, 5′-AAG AGG CCG AGA 3934 submit your manuscript | www.dovepress.com OncoTargets and Therapy 2019:12 DovePress Dovepress Yi et al CTT GTG AA-3′, human-N-cadherin-AS, 5′-CAC TGG plate at l×106/well, and siRNA were transfected with GGA TAA GGG AAG GT-3′; human-vimentin-S, 5′-GAG Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) AAC TTT GCC GTT GAA GC-3′, human-vimentin-AS, 5′- when the cell abundance was up to 70%~80%. The final GCT TCC TGT AGG TGG CAA TC-3′;human-CAPG-S, concentration of siRNA transfection was 200 nm/L. After 5′-GGG GAC TCC TAC CTA GTG CTG-3′, human-CAPG transfection for 48 h, the knockdown efficiency was eval- -AS, 5′-CAC CAC CTT CCT GGT ACT TGA-3′.RT- uated by Western blot analysis. qPCR was conducted using SYBR Premix Ex Taq™ (Takara, Dalian, China) at 95 °C for 30 sec, followed by Construction and transfection of 40 cycles of 95 °C for 5 sec and 60 °C for 34 sec in the ABI overexpression vectors NSD3 StepOnePlus Real-time PCR system. The relative fold A pair of primers for the known NSD3 coding sequences was changes in mRNA expression were calculated using the designed as mentioned earlier.