NEUROBIOLOGY & PHYSIOLOGY ANIMAL CELLS AND SYSTEMS 2020, VOL. 24, NO. 2, 107–113 https://doi.org/10.1080/19768354.2020.1736623 Knockdown of LncRNAZFAS1 suppresses cell proliferation and metastasis in non- small cell lung cancer Yong Zhou, Xiao-Wei Hu, Si-Jia Yang and Zhe Yu Department of Respiratory Medicine, HwaMei Hospital, University Of Chinese Academy Of Sciences, Ningbo, People’s Republic of China ABSTRACT ARTICLE HISTORY Received 8 February 2020 To evaluate the eﬀects of LncRNAZFAS1 on cell proliferation and tumor metastasis in non-small cell Revised 24 February 2020 lung cancer (NSCLC), we detected the expression level of LncRNAZFAS1 in NSCLC-related tissues Accepted 25 February 2020 and cells. qRT-PCR results revealed that LncRNAZFAS1 in tumor tissues was signiﬁcantly higher than that in normal lung tissue, especially signiﬁcantly up-regulated in stage III / IV and in KEYWORDS metastatic NSCLC tissues. LncRNAZFAS1 expression was dramatically up-regulated in 4 NSCLC- LncRNAZFAS1; non-small cell related cells (A549, SPC-A1, SK-MES-1, and NCI-H1299), with having the highest expression level lung cancer (NSCLC); in A549 cells. Furthermore, we implemented a knockdown of LncRNAZFAS1 in A549 cells, and proliferation and metastasis; the results of CCK8 and Transwell assays suggested that knockdown of LncRNAZFAS1 biomarker signiﬁcantly inhibited NSCLC cell proliferation and metastasis. Next, we constructed a tumor xenograft model to evaluate the eﬀect of LncRNAZFAS1 on the NSCLC cell proliferation in vivo. The results indicated that knockdown of LncRNAZFAS1 dramatically inhibited A549 cells proliferation and repressed tumor growth. Additionally, knockdown of LncRNAZFAS1 drastically weakened the expressions of MMP2, MMP9 and Bcl-2 proteins, whereas noticeably strengthened the expression of BAX protein. Our results altogether suggest that knockdown of LncRNAZFAS1 has a negative eﬀect on the proliferation and metastasis of NSCLC cell, which implying LncRNAZFAS1 is a potential unfavorable biomarker in patients with NSCLC. Introduction biomarkers is still a pressing issue to develop targeted Based on the estimates of GLOBOCAN 2018 (Bray et al. therapy or immunotherapy (Ettinger et al. 2018). 2018), lung cancer (LC) remains the leading cause of Numerous previous studies have illuminated that non- cancer-associated death worldwide, with 2.1 million coding RNAs (ncRNAs), such as long non-coding RNAs new incidence cases and 1.8 million deaths predicted (LncRNAs), microRNAs (miRNAs), small nucleolar RNAs in 2018. Among males, there is the highest incidence (snoRNAs), are emerging as a critical regulator or key rate in China (Bray et al. 2018). Non-small-cell lung elements of cell proliferation and metastasis in human cancer (NSCLC) is the most common type of lung non-small-cell lung cancer (Ji et al. 2003; Liao et al. cancer (Ettinger et al. 2013), with occupying over 85% 2010; Tang et al. 2017). Over the past decades, multiple of LC cases (Ettinger et al. 2010). More generally, an LncRNAs (deﬁned as > 200 nucleotides in length) have increasing body of studies points to the fact that most been annotated and identiﬁed key roles in cell cycle NSCLC patients are already in advanced or metastatic and regulating gene expression in cancers (Gutschner NSCLC stage at the time of initial diagnosis (Fossella and Diederichs 2012; Spizzo et al. 2012; Shi et al. 2013), et al. 2000; Giaccone et al. 2004; Johnson et al. 2004; such as LncRNA HOTAIR (Gupta et al. 2010), LncRNA Thatcher et al. 2005), resulting in a lower 5-year survival MALAT-1 (Ji et al. 2003), LncRNA AFAP1-AS1 (Leng et al. rate (< 5%); even though patients with NSCLC are diag- 2018), LncRNA NEAT1 (Sun et al. 2016), LncRNA TUG1 nosed at a very early stage, the 5-year survival remains (Zhang et al. 2014). Furthermore, a comprehensive assess- lower than 50% (Detterbeck et al. 2009; Velcheti et al. ment suggested that the dysregulation of lncRNAs has 2014). Despite the improvement of treatments, as a con- increasingly become an endogenous force in the for- sequence, the latest NCCN Clinical Practice Guidelines for mation of human cancers (Shi et al. 2013; Leng et al. 2018). human non-small-cell lung cancer recommends that Recent reports have been investigated that long-term aiming at key predictive biomarkers and prognostic non-coding RNA zinc ﬁnger antisense 1 (LncRNAZFAS1) CONTACT Zhe Yu email@example.com Department of Respiratory Medicine, HwaMei Hospital, University Of Chinese Academy Of Sciences, No. 41, Northwestern Street, Ningbo, Zhejiang Province, 315010, People’s Republic of China This article has been republished with minor changes. These changes do not impact the academic content of the article. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 108 Y. ZHOU ET AL. is overexpressed in nasopharyngeal carcinoma (NPC), as added to the reaction of 20 μL. SYBR® Green Master well as up-regulated in colonic cancer (Fang et al. 2017; Mix Kit (Takara, China) was executed to perform qRT- Chen et al. 2018). However, there is little evidence as to PCR, following manufacturer’s protocol. The primer how LncRNAZFAS1 inﬂuence the cell proliferation and sequences of LncRNAZFAS1 were as follow: primer-F: metastasis in NSCLC. Therefore, in this study, the aims 5’-CCGGAGTGTGGTACTTCTCC-3’; primer-R: 5’-CCA- are to investigate the expression of LncRNAZFAS1 in GAGGTCTCCAACGAAGA-3’. The standard cycling NSCLC-related tissues and cells, to determine the role program was: 5 min at 95°C, followed by 40 cycles of of LncRNAZFAS1 on NSCLC cell proliferation and metas- 30 s at 95°C, 45 s at 65°C. GAPDH was used to normalize tasis, and to evaluate the predictive value in diagnosis or the expression (primer-F: 5’-GGTGGTCTCCTCTGACTT- therapy of NSCLC. CAA-3’; primer-R: 5’-GTTGCTGTAGCCAAATTCGTTGT-3’). The relative mRNA expression level in each group was −ΔΔCT analyzed by the 2 method. Every sample was per- Material and methods formed in triplicate. Patients and samples Lung cancer tissues and adjacent normal tissues were Knockdown of LncRNAZFAS1 sampled from 77 patients with NSCLS (46 males and 31 A lentiviral system was used to knock down the females, aged 35–75 years, with a median age of 56.8 LncRNAZFAS1 (sh-LncRNAZFAS1). HEK-293T cells (2 × years) in HwaMei Hospital, University Of Chinese 10 cells/dish) were seeded in cell culture dishes and Academy Of Sciences (China) from November 2015 to cultured for 1 day before being subjected to a lentivirus February 2018. All patients were diagnosed as NSCLC packaging system. Lenti-Pac HIV Mix (5 μL) and 2.5 μg based on histopathological features and hematology plasmid were mixed and added into 200 μLofserum- examination and categorized into diﬀerent stages free DMEM medium, then added into 15 μLofEndoFec- through the TNM staging system of malignant tumors tin Lenti, ﬁnally transferred into HEK-293T cells after after surgery. According to the TNM classiﬁcation, 17 incubated for 20 min at room temperature. The cases were in stage I, 19 cases were in stage II, 24 cases medium was replaced after transfection for 8 h. 20 μL were in stage III, 17 were cases in stage IV; 33 cases of TiterBoost Reagent was added into the medium. Len- had lymph node metastasis and 44 cases were free tivirus was collected after 2 days. 200 μLofvirus sol- metastasis. All patients had detailed clinical data and ution and 1.5 mL of complete medium were added to had never received any therapy before diagnosis. All col- A549 cells (6-well plate), and after further culture for 2 lected samples were immediately snap-frozen in liquid days, 0.5 μg/mL of puromycin was added to screen nitrogen and stored at −80°C until required. stable cell lines. A549 cells transfected with an empty lentiviral vector were used as a negative control (NC) Cell culture group, and unmanipulated A549 cells were used as a blank control (BC) group. The human NSCLC-related cell lines (SPC-A1, A549, SK- MES-1, NCI-H1299) and normal human bronchial epi- thelial cells (16HBE) were purchased from American CCK8 assay Type Culture Collection (ATCC) Bank. 16HBE cells and The cell viability of NSCLC was tested by cell counting kit- NCI-H1299 cells were cultured in Roswell Park Memorial 8 (CCK8) (MSK Bio. Inc.) A549 cells transfected with sh- Institute 1640 (RPMI 1640) medium (Gibco, USA) contain- LncRNAZFAS1 (5 × 10 cells/well) were seeded in 96- ing 10% fetal bovine serum (FBS) (Gibco, USA). A549 well plates and were cultured at 1, 2 days, and 3 days cells, SPC-A1 cells, and SK-MES-1 cells were cultured in for measuring. After cell adherence, 100 μL CCK-8 sol- DMEM medium (Thermo Fisher Scientiﬁc, Inc.) contain- ution (10%) was added in each well and incubated for ing 10% FBS. The above cells were cultured in humidiﬁed 1.5 h at 37°C. The optical density (OD) values at 450 nm air with 5% CO at 37°C based on the conventional cell were measured as the positive index of cell viability by culture method. a microplate reader. RNA extraction and qRT-RNA Transwell invasion assay Total RNA was extracted from tissue samples according Pre-cooled DMEM medium and Matrigel were to the manufacturer’s protocol of TRIZOL RNA extraction thoroughly mixed in a ratio of 1:1; 0.1 mL of the kit (TIANGEN). AMV reverse transcription kit was used to mixture was uniformly was evenly added to the reverse transcribe RNA, in which 2 μg total RNA was bottom of the upper chamber; and incubated for 4 h. ANIMAL CELLS AND SYSTEMS 109 0.2 mL A549 cells of each treatment (2.5 × 10 cells/well) LncRNAZFAS1 promoted cell proliferation and in the phase of logarithmic growth were added to the metastasis of NSCLC upper chamber, and 0.5 mL DMEM medium (containing To determine the eﬀect of high expression of LncRNAZ- 10% FBS) was added to the lower chamber. After 2 FAS1 on cell proliferation and metastasis of NSCLC, we days of culture, crystal violet staining was performed, implemented the knockdown of LncRNAZFAS1 in A549 and 5 randomly ﬁelds were selected to count. cells (P < 0.001, Figure 2(A)). Next, the CCK8 assay showed that knockdown of LncRNAZFAS1 signiﬁcantly inhibited A549 cell proliferation (P < 0.001, Figure 2(B)). Western blotting The results of Transwell invasion assay (Figure 2(C)) The collected tumor tissues were added to liquid nitro- showed that the number of migrated cells in A549 cells gen for grinding, and the supernatant was collected by transfected with sh-LncRNAZFAS1 was signiﬁcantly centrifugation at 4°C for 30 min (12,000 rpm/min). The lower than that in the negative control group (NC protein concentration of each group was adjusted by group) and the blank control group (BC group) (P < the BCA method. A mixture of protein solution and 0.001). loading buﬀer (1:1 ratio) was used for denaturation, with boiling at 100°C for 5 min. 30 μL protein samples were subjected to SDS-PAGE electrophoresis. Knockdown of LncRNAZFAS1 suppressed NSCLC Consequently, proteins were transferred onto a PVDF cell proliferation in vivo membrane. Corresponding primary antibody was incu- To evaluate the eﬀect of LncRNAZFAS1 on the NSCLC cell bated for 2 h at room temperature, and then the sec- proliferation in vivo, we constructed a tumor xenograft ondary antibody was also incubated for 2 h used at model using the A549 cell transfected with sh-LncRNAZ- room temperature. Finally, the membrane was FAS1. The rate of tumor growth was signiﬁcantly down- detected using an enhanced chemiluminescent detec- regulated when LncRNAZFAS1 knocked down, with sig- tion system. niﬁcantly decreasing tumor volume and tumor weight (P < 0.001; Figure 3(A and B)). To further detect prolifer- ation- and metastasis-associated proteins, WB results Statistical analysis revealed that knockdown of LncRNAZFAS1 signiﬁcantly repressed MMP2, MMP9, and Bcl-2, whereas drastically Each treatment has 3 replicates for calculating the up-regulated BAX protein (P < 0.001; Figure 3(C)). average. The diﬀerence comparison was performed by the SNK-Q test in SPSS v23.0 software. All data were expressed as mean ± SD (N = 3). P < 0.05 was considered Discussion as a statistically signiﬁcant diﬀerence. The LncRNAs have been reported to become a thera- peutically eﬀective predictive biomarker or prognostic Results biomarker in the process of cancer diagnosis, prognosis and metastasis (Spizzo et al. 2012; Qiu et al. 2013; Shi LncRNAZFAS1 was up-regulated in NSCLC-related et al. 2013). To address the knowledge gap of how tissue and cells LncRNAZFAS1 inﬂuence NSCLC cell proliferation and To investigate the expression of LncRNAZFAS1, qRT-PCR metastasis, in this study, we described the expression results revealed that LncRNAZFAS1 was signiﬁcantly up- level of LncRNAZFAS1 in NSCLC. qRT-PCR results regulated in tumor tissues, with 2.92-fold higher than revealed that LncRNAZFAS1 was signiﬁcantly up-regu- that in normal tissues (P < 0.001, Figure 1(A)); LncRNAZ- lated in NSCLC-related tissues and cells, compared with FAS1 expression in stage III/IV NSCLC was signiﬁcantly relative normal tissues and cells. Similar results were higher than that in stage I/II NSCLC (P < 0.001; Figure 1 investigated in nasopharyngeal carcinoma, colorectal (B)); LncRNAZFAS1 expression in metastatic patients cancer (CRC) and gastric cancer (Thorenoor et al. 2016; was signiﬁcantly higher than that in non-metastatic Fang et al. 2017; Chen et al. 2018; Xie et al. 2018;Xu patients (P < 0.001; Figure 1(C)); compared to the et al. 2018). To indicate the relationship between the normal 16HBE cells, LncRNAZFAS1 expression was sig- expression level of LncRNAZFAS1 and tumor stage and niﬁcantly up-regulated in 4 NSCLC-related cells (A549, metastasis, we found that LncRNAZFAS1 was signiﬁ- SPC-A1, SK-MES-1, and NCI-H1299) (P < 0.001), with cantly overexpressed in stage III / IV and in metastatic LncRNAZFAS1 having the highest expression level in NSCLC tissues, further indicating that elevated LncRNAZ- A549 cells (Figure 1(D)). FAS1 was remarkably correlated with TNM stage and 110 Y. ZHOU ET AL. Figure 1. LncRNAZFAS1 was highly expressed in NSCLC and tumor-related cells lines. (A) expression of LncRNAZFAS1 in lung cancer tissues was signiﬁcantly higher than that in normal lung tissues (N = 77); the expressive variation of LncRNAZFAS1 between NSCLC staging (I/II: 36; III/IV: 41) (B) and metastasis (lymph node metastasis: 33; free metastasis: 44) (C); (D) LncRNAZFAS1 dramatically expressed in 4 diﬀerent NSCLC-related cell lines (N = 6). Data were shown as mean ± SD, *** P < 0.001. lymph node metastasis and may promote the pro- we implemented a knockdown of LncRNAZFAS1 in gression and metastasis of NSCLC. A549 cells. Results indicated that NSCLC cell proliferation To assess the eﬀect of LncRNAZFAS1 on cell prolifer- and metastasis processes were dramatically inhibited ation and metastasis in NSCLC cell, on the other hand, when LncRNAZFAS1 was knocked down in A549 cells. Figure 2. The eﬀect of LncRNAZFAS1 on cell proliferation and metastasis of NSCLC. (A) Construction of LncRNAZFAS1 knockdown (N = 6); (B) The cell viability in A549 cells transfected with sh-LncRNAZFAS1 were measured by CCK-8 assay at 1, 2 days, and 3 days (N =6); (C) The metastasis of A549 cells transfected with sh-LncRNAZFAS1 were measured by Transwell invasion assay (N = 6). shRNA: A549 cells transfected with sh-LncRNAZFAS1; NC: A549 cells transfected with an empty lentiviral vector; BC: unmanipulated A549 cells. Data were shown as mean ± SD, **P< 0.01, *** P < 0.001. ANIMAL CELLS AND SYSTEMS 111 Figure 3. The eﬀect of LncRNAZFAS1 on the NSCLC cell proliferation in vivo. Knockdown of LncRNAZFAS1 suppressed NSCLC cell pro- liferation, with decreasing tumor volume (A) and tumor weight (B) in a tumor xenograft model (N = 20). (C) The knockdown of LncRNAZFAS1 signiﬁcantly down-regulated the proteins MMP2, MMP9, and Bcl-2, whereas up-regulated the protein Bax (N = 20). shRNA: A549 cells transfected with sh-LncRNAZFAS1; NC: A549 cells transfected with an empty lentiviral vector; BC: unmanipulated A549 cells. Data were shown as mean ± SD, ** P < 0.01, *** P < 0.001. ## P < 0.01, ### P < 0.001. However, the physical, chemical and growth conditions LncRNAZFAS1 suppressed malignancies (e.g. gastric of NSCLC cells in vitro diﬀer from that in vivo, which cancer, nasopharyngeal carcinoma) via blocking Wnt/β- may aﬀect the cell proliferation and metastasis. Next, catenin signaling pathway (Chen et al. 2018; Xu et al. we constructed a tumor xenograft model to evaluate 2018). The stability of β-catenin was regulated by a the eﬀect of LncRNAZFAS1 on the NSCLC cell prolifer- destruction complex (DC) in which tumor suppressor ation in vivo. Results indicated that the growth of A549 protein Axin interacted with tumor suppressor APC, β- cells was dramatically inhibited after reducing the catenin and serine-threonine kinases (CK1α/δ and expression of LncRNAZFAS1. Additionally, knockdown GSK3α/β) (Nusse and Clevers 2017). On the other hand, of LncRNAZFAS1 was drastically down-regulated the some studies illuminated that LncRNAZFAS1 can regu- expressions of matrix metalloproteinases MMP2, MMP9 late the intracellular processes via some speciﬁc micro- and apoptosis-regulating protein Bcl-2 proteins, but sig- RNAs (e.g. miR-27a, miR-484) (Xie et al. 2018;Yeetal. niﬁcantly up-regulated the expression of pro-apoptotic 2018). However, some evidence have already indicated regulator BAX. It implicated that the low expression of that miR-27a regulates cell proliferation and invasion LncRNAZFAS1 weakened the metastasis of NSCLC. by targeting the SFRP1 gene through activating Wnt/β- Because the low expression of LncRNAZFAS1 can block catenin signaling pathway (Guo et al. 2014; Kong et al. the G0 / G1 phase of the cell cycle and activate cell apop- 2017; Wu et al. 2017). Next, we will further determine tosis (Chen et al. 2018). These observations further imply whether LncRNAZFAS1 promotes cell proliferation and that high-expression of LncRNAZFAS1 in primary tumors migration in NSCLC via Wnt/β-catenin signaling pathway. is signiﬁcantly associated with tumor growth and metastasis. The Wnt signaling pathway emerged as a fundamen- Conclusion tal cell growth control pathway control multiple pro- cesses of cell regulatory, such as inducing cells to In conclusion, LncRNAZFAS1 signiﬁcantly was overex- proliferate and transcriptional activation, activating cell- pressed in NSCLC, and knockdown of LncRNAZFAS1 polarizing intracellular signaling cascades, regulating inhibited cell proliferation and metastasis. All the cell apoptosis (Chen et al. 2001; Schuijers et al. 2014; ﬁndings imply that LncRNAZFAS1 is expected to Loh et al. 2016; Nusse and Clevers 2017), etc. Recent become a potential predictive biomarker for patients studies have already illustrated that knockdown of with advanced and metastasis NSCLC. 112 Y. ZHOU ET AL. previously treated with platinum-containing chemotherapy Acknowledgement regimens. J Clin Oncol. 18:2354–2362. None. Giaccone G, Herbst RS, Manegold C, Scagliotti G, Rosell R, Miller V, Natale RB, Schiller JH, von Pawel J, Pluzanska A, et al. 2004. Geﬁtinib in combination with gemcitabine and cisplatin in Disclosure statement advanced non–small-cell lung cancer: a phase III trial— INTACT 1. J Clin Oncol. 22:777–784. No potential conﬂict of interest was reported by the author(s). Guo D, Li Q, Lv Q, Wei Q, Cao S, Gu J, Zheng SG. 2014. MiR-27a targets sFRP1 in hFOB cells to regulate proliferation, apopto- sis and diﬀerentiation. PloS one. 9:e91354. Funding Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, Tsai This study was supported by Medical Health Science and Tech- M-C, Hung T, Argani P, Rinn JL, et al. 2010. Long non-coding nology Project of Zhejiang Provincial Health Commission (No. RNA HOTAIR reprograms chromatin state to promote cancer 2018268491), Ningbo Health Branding Subject Fund (No. metastasis. Nature. 464:1071. 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Animal Cells and Systems
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Published: Mar 3, 2020
Keywords: LncRNAZFAS1; non-small cell lung cancer (NSCLC); proliferation and metastasis; biomarker