Med Oncol (2015) 32:136 DOI 10.1007/s12032-015-0594-y
ORIGINAL PAPER
Knockdown of HMGN5 suppresses the viability and invasion of human urothelial bladder cancer 5637 cells in vitro and in vivo Yu Gan1 • Jing Tan1 • Jianfu Yang1 • Yihong Zhou1 • Yingbo Dai1 Leye He1 • Kun Yao1 • Yuxin Tang1
•
Received: 14 March 2015 / Accepted: 16 March 2015 / Published online: 22 March 2015 Ó Springer Science+Business Media New York 2015
Abstract The high-mobility group nucleosome-binding domain 5 (HMGN5) is a new and typical member of HMGN protein family. Numerous studies confirmed that HMGN5 was highly expressed in several kinds of malignant tumors, but its role in cancer progression of urothelial bladder cancer (UBC) has not been fully clarified. This study aimed to further investigate the oncogenic role of HMGN5 in UBC 5637 cells employing in vitro and in vivo models and explored the mechanism. RNA interference was used to down-regulate HMGN5 expression in 5637 cells by a shRNA expression lentiviral vector. Then cell viability, apoptosis and cell cycle distribution, invasion were detected by MTT assay, flow cytometry and transwell assay, respectively. Tumor growth was also evaluated in nude mice. As a result, successful transfection was confirmed using fluorescence microscopy and HMGN5 was efficiently inhibited. HMGN5 knockdown suppressed invasion induced G1/S cell cycle arrest but not apoptosis and thus contributed to decreased cell viability in UBC 5637 cells. Consistent with the cell cycle arrest, the protein expression levels of cyclin D1 were decreased. In vivo study further showed that HMGN5 knockdown affected the tumorigenesis of 5637 cells in nude mice. Western blot also demonstrated that the expression of E-cadherin and VEGFC was decreased in 5637 cells depleted of HMGN5. In conclusion, we provide both in vivo and in vitro evidence that HMGN5 contribute to the growth and invasion of UBC
& Kun Yao [email protected] 1
Department of Urology, The Third Xiangya Hospital of Central South University, 138 Tongzipo Road, Changsha, Hunan 410013, People’s Republic of China
5637 cell line and HMGN5 could be exploited as a target for therapy in UBC. Keywords HMGN5 Urothelial bladder cancer Cell viability Invasion RNA interference
Introduction Urothelial bladder cancer (UBC) is the seventh most common cancer in men and ranks the 17th in women worldwide, with an estimated 110,500 male and 7000 female cases newly diagnosed in the European Union each year [1]. Although nearly 75 % of firstly diagnosed UBCs are noninvasive, it is still a real challenge for urologists to treat them, due to the propensity for disease recurrence and progression [2]. Moreover, radical cystectomy, chemotherapy and radiotherapy for muscle-invasive or metastatic UBCs always have a significantly negative impact on the quality of life and barely bring satisfactory prognosis [3]. So it is important to further understand the pathogenesis of UBC so as to develop effective methods for the early diagnosis and treatment of UBC. High-mobility group N (HMGN) family was previously considered as chromatin architectural proteins only working on regulating the chromatin structure. But latest research indicates that these proteins may have a crucial role in repair of DNA lesions and transcriptional control [4, 5]. Since accumulated DNA lesions which possibly result in genetic mutations and chromosomal aberrations, and aberrant transcriptional level of cancer-related genes have been proved as causes of malignant tumors, it is reasonable to link HMGNs with tumorigenesis [6, 7].
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The high-mobility group nucleosome-binding domain 5 (HMGN5) is a novel and featured member of HMGN protein family with ubiquitous expression in mouse and human tissues [8]. Early studies showed up-regulation of the HMGN5 protein was indentified in certain kinds of malignant tumors, such as prostate cancer, clear cell renal cell carcinoma, squamous cell carcinoma and golima, indicating the carcinogenic role of this protein [9–12]. Recently, Wahafu et al. [13] reported that the HMGN5 protein was over-expressed in human bladder cancer cell lines and tumor tissues, and HMGN5 expression was correlated with the tumor grade and pathologic stage. Though knockdown of HMGN5 via transient transfection was proved to reduce the viability of EJ cells through inducing G2 phase arrest in their study, the contribution of HMGN5 to the invasion ability was speculated. What’s more, further molecular mechanism and in vivo study are also critical to elucidate the role of HMGN5 in cancer progression of UBC. Therefore, in the present study, we investigated the effects of lentivirus mediated HMGN5 knockdown on the growth and invasion of UBC 5637 cells and in vivo tumor growth in nude mice. Our results demonstrated that HMGN5 knockdown induced G1 phase arrest but not apoptosis and thereby reduced the viability of 5637 cells; additionally, the invasion potential was inhibited via upregulating VEGF-C and down-regulating E-cadherin, and tumor growth in vivo was also suppressed.
Methods Cell culture Human UBC 5637 cell line was procured from Yingrun Biotechnologies (Changsha, China). All cells were cultured in RPIM-1640 medium (InvitrogenTM Life Technologies, NY, USA) supplemented with 10 % fetal bovine serum (Invitrogen) under the condition of 37 °C, 5 % CO2 in a standard humidified incubator. Cells in the logarithmic phase were used for the experiments. Lentivirus RNAi construct and transfection A 21-bp fragment within human HMGN5 cDNA (NM_030763) was selected as the target for HMGN5 siRNA, as follows: 50 -GTTGTTGAAGAAGACTACAAT-30 . The other fragment (50 -GACTTCATAAGGCGCATGC-30 ) had no significant homology to human gene sequences and was chosen as siRNA control. Short hairpin RNA (shRNA) fragments were hybridized with synthesized sense and antisense oligonucleotides. The shRNA fragments were then cloned to pYr-Lvsh to construct the lentiviral vectors. The
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positive clones were verified by restriction mapping and direct DNA sequencing. Then the recombinant lentivirus was packaged and concentrated by Yingrun Biotechnologies, and the final virus titer was determined. Four hours before transfection, 5637 cells were placed in serum-free media. Then the cells were infected with the recombinant lentivirus at a multiplicity of infection (MOI) of 50 according to the manufacturer’s protocol. And 1 lg/ ml puromycin (Sigma-Aldrich, St. Louis, MO, USA) could be used for screening positively stable transfectants if needed. Quantitative real-time PCR assay Total RNA was extracted from the cells with Trizol reagent (Invitrogen) according to the manufacturer’s instruction at 72 h post-transfection. The concentration and purity of the RNA samples were determined at 260 nm. First-strand cDNA was generated through reverse transcription via RevertAidTM Reverse Transcriptase (Thermo ScientificTM Life Technologies, NY, USA). The specific primers were synthesized by Sangon (Shanghai, China) and as follows: HMGN5, 50 -CAGGTCAAGGTGATATGAG GCA-30 (forward) and 50 -GCTTGGGCACTTGTATCTATGT-30 (reverse); GAPDH, 50 -TGCACCACCAACTGCTTAGC-30 (forward) and 50 -GGCATGGACTGTGGTCATGA G-30 (reverse). THUNDERBIRDTM SYBRÒ qPCR Mix (Toyobo, Osaka, Japan) was used for real-time PCR, and the reaction system was 20 ll. Thermal cycling was started with a denaturation step for 5 min, followed by 43 cycles done in three steps: 30 s at 95 °C, 30 s at 58 °C and 20 s at 72 °C. GAPDH was used as the internal control, and gene expression was relatively quantified using 2-DDCT method [14]. Western blotting The cells were harvested for the analysis of protein expression at 72 h post-transfection and lysed on ice by RIPA lysis buffer. Protein concentration of lysate was determined by the bicinchoninic acid assay (Beyotime Biotechnology, Shanghai, China). Then protein samples (40 lg) were separated by SDS-PAGE and subsequently transferred onto a PVDF membrane. After blocked with nonfat milk in TBST, the membrane was incubated with primary antibodies against HMGN5 (Abcam, MA, USA), cyclin D1 (Santa Cruz, CA, USA), VEGF-C, E-cadherin and b-actin (Cell Signaling Technology, MA, USA) and then incubated with HRP-conjugated secondary antibody (Santa Cruz). Immunoreactivity signals were developed by ECL reagent (GE Healthcare Bioscience, NJ, USA) and recorded by a fluorescence detection system.
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Cell viability assay The cells were seeded in 96-well plates at a density of 1 9 103/well in a volume of 100 ll and grown overnight. Four hours before transfection, the cells were changed to serum-free medium and transfected with lentivirus. After incubated for 48, 72 or 96 h, 20 ll MTT (5 mg/ml) was added to each well ,then cell plates were put in the incubator for 4 h, and next 150 ll DMSO was added to each well to lyse the cells. Absorbance was measured at 490 nm. Cell cycle analysis The cells were seeded in 25-cm2 flasks. After growing to sufficient confluence, the cells were changed to serum-free medium for 24 h and transfected with lentivirus. The cells were cultured for 72 h and then harvested by gentle trypsinization, extensively washed with cold PBS and collected by centrifugation. Next the cells were resuspended in 0.5 ml PBS and fixed in 1 ml 75 % cold ethanol at 4 °C overnight. After brief centrifugation, the cells were washed once with PBS and incubated in 37 °C for 1 h in 1 ml PBS with 5 ll RNase (10 mg/ml) and then filtered and incubated in the dark for 30 min with 100 lg/ml of propidium iodide. For each tube, 2 9 104 cells were immediately analyzed using a FACS Canto II instrument (BD, CA, USA) and further analyzed using the program with it.
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the upper chamber of each transwell insert (pore size 8 lm, Corning, NY, USA), and then they were incubated at 37 °C for 5 h to make the Matrigel changed from liquid form to gelatum. After hydration of the Matrigel with serum-free medium, 100 ll cells with a density of 5 9 104/ml were seeded in upper chamber; meanwhile, 600 ll medium containing 10 % FBS was filled into the lower chamber. The cells were then cultured at 37 °C for 24 h to allow cells migration. In the end, the noninvasive cells in the upper chamber were gently wiped out with a cotton swab, and the invasive cells attached to the lower chamber were fixed with 4 % paraformaldehyde and stained with 0.1 % crystal violet. Five visual fields were selected randomly, and the cells were counted for each insert and photographed under a light microscope at 2009 magnification. Xenograft The present animal experiment was approved by the Animal Ethics Committee of The Third Xiangya Hospital. Female BALB/c-nu mice (4–5 weeks old, weighed 14–16 g) were maintained in a germ-free environment for 1 week in the animal facility. Then HMGN5 knockdown or siRNA control 5637 cells (4 9 106 in 200 ll medium) were injected subcutaneously in the armpit area. Tumor parameters were measured every week, and tumor volume was calculated by length 9 width2 9 0.5. All the mice were killed after 5 weeks.
Cell apoptosis analysis Data processing An Annexin V-FITC Apoptosis Detection Kit (KeyGEN, Nanjing, China) was used following the manufacturer’s instruction. The cells were seeded in the six-well plates and cultured in the incubator for sufficient confluence. Four hours before transfection, the cells were changed to serumfree medium and then transfected with lentivirus. After 72 h of incubation, the cells were gently trypsinized and washed twice with cold PBS. Then the cells were resuspended in 500 ll 1 9 binding buffer as a density of 1 9 106/ml, and then 100 ll cell suspension was collected, stained with 5 ll of Annexin V-FITC (25 lg/ml) and 5 ll propidium iodide and then incubated on ice in the dark for 15 min. Every sample was diluted with 400 ll PBS and filtered and immediately analyzed using a FACS Canto II instrument (BD).
All the experiments were independently repeated in triplicate, and data were collected. Data were expressed as mean ± standard deviation (mean ± SD) and analyzed for statistical significance using GraphPad Prism 5.01 software (GraphPad Software, CA, USA). Differences among the means of multiple groups were compared based on ANOVA, followed by a Newman–Keuls multiple comparison test. T test was used to compare the difference between two groups. A value of p \ 0.05 was considered as statistically significant.
Results
Cell invasion assay
HMGN5 knockdown decreases the viability of 5637 cells
The invasion capacity of the cells was measured using transwell chamber assay at 72 h post-transfection. The Matrigel (BD) was put into a 4 °C refrigerator overnight for melting. The next day, the Matrigel was diluted by 1:5 with serum-free medium, and 100 ll Matrigel was added to
A total of 5637 cells are derived from a grade II UBC patient, and this cell line is a suitable transfection host [15]. Previous study already identified HMGN5 expression in 5637 cells was higher than normal human urinary tract epithelial cells [13]. Hence, to further investigate the role of HMGN5 in the
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cell biology of UBC cells, we employed 5637 cell line as a model and the loss of function method. A total of 5637 cells were transfected with HMGN5 siRNA or scramble siRNA as RNAi control. Successful transfection of 5637 cells was determined by detecting GFP expression using fluorescence microscopy (Fig. 1). The results showed that lentivirus short hairpin constructs against HMGN5 were efficient and specific in the knockdown of the gene in 5637 cells, with the inhibitory efficiency at mRNA level was 72 ± 5 % based on RT-PCR and at protein level was 60 ± 2 % based on Western blot (Fig. 1). Fig. 1 (a) Confirm the successful transfection by detecting GFP expression using fluorescence microscopy. The transfection efficiency is above 80 % for both two transfection groups (magnification, 9200). (b) Knockdown of HMGN5 in 5637 cells. A total of 5637 cells are transfected with HMGN5 siRNA or scrambled siRNA for 72 h. Quantitative real-time PCR confirmed the HMGN5 mRNA level in the 5637 cells with HMGN5 knockdown is significantly decreased (GAPDH served as the internal control). (c) Similar results are also observed in the protein level, which is confirmed by Western blot analysis (b-actin served as the internal control). Results are expressed as mean ± SD, n = 3. **p \ 0.01 compared to the 5637 cells with scrambled siRNA
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Firstly, cell viability was evaluated by MTT assay and the results showed that HMGN5 knockdown significantly suppressed the viability of 5673 cells over 72 h period (Fig. 2). To ascertain whether this is due to the cell cycle arrest or increased apoptosis, we then performed flow cytometry analysis, and we observed no significant increase in apoptosis in 5637 cells transfected with HMGN5 siRNA (Fig. 2), which was similar to previous report from Wahafu et al. [13]. However, in the cell cycle analysis, we identified an abundant increase in G1 phase fraction of the cells with HMGN5 knockdown, which indicated the G1 phase
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Fig. 2 (a) Detection of cell viability by MTT assay. Cell viability is expressed as the absorbance values. The viability of the 5637 cells with HMGN5 knockdown is suppressed over 72 h after transfection. Results are expressed as mean ± SD, n = 3, *p \ 0.05, **p \ 0.01 compared to the RNAi control 5637 cells. (b) Detection of cell apoptosis by flow cytometry. Statistical analysis of percentage of apoptotic cells failed to show significantly different at 72 h posttransfection among the three groups (p [ 0.05). HMGN5 knockdown induced G1/S phase arrest in 5637 cells at 72 h post-transfection.
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(c) a Cell cycle distribution is performed by flow cytometry. The percentage of cells at G1 phase is moderately increased in the 5367 cells with HMGN5 knockdown compared to that in the 5637 cells with scrambled siRNA (p \ 0.01). b Cyclin D1 is a sensitive checkpoint of G1 phase in cell cycle. Western blot analysis confirmed the expression of cyclin D1 in the 5367 cells with HMGN5 knockdown is moderately suppressed (p \ 0.05). b-Actin served as the internal control. Results are expressed as mean ± SD, n = 3, **p \ 0.01
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arrest in these cells (Fig. 2). Cyclin D1 is one of the crucial checkpoints of G1/S transition [16]. To further confirm HMGN5 modulates cell cycle progression of 5637 cells, we examined the level of cyclin D1 by Western blot analysis and found it was also significantly decreased in the cells with HMGN5 knockdown (Fig. 2). Collectively, these results suggest that HMGN5 might modulate the G1 phase of cell cycle but not apoptosis and contribute to the viability of 5637 cells.
Fig. 3 (a) Test of cell invasiveness by transwell assay. The cells penetrating the filter membrane at 72 h posttransfection are stained with crystal violet and are shown as blue under a light microscope (magnification, 9 200). a Blank control. b RNAi control. c HMGN5-RNAi. d The quantitative results showed a greatly reduced invasiveness of the 5637 cells with HMGN5 knockdown compared to that of RNAi control cells. (b) Western blot for HMGN5, E-cadherin, VEGF-C and b-actin at 72 h post-transfection. HMGN5 knockdown inhibited the expression of E-cadherin (p \ 0.01) and VEGF-C (p \ 0.01) in 5637 cells. bActin served as the internal control
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HMGN5 knockdown inhibits the invasion of 5637 cells Next we utilize transwell invasion assay to confirm the previous speculation in the role of HMGN5 in UBC cells invasion. The filter was stained with crystal violet and inspected under the microscope (Fig. 3); we found HMGN5 siRNA significantly inhibited the invasiveness of 5637 cells. The number of cells penetrating the Matrigel was
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20.7 ± 4.2 in HMGN5 siRNA group versus 65.4 ± 24.5 in blank control group (p \ 0.01; Fig. 3). E-cadherin, a calcium-dependent transmembrane glycoprotein, is required for cell–cell adhesion and viewed as a key tumor suppressor related with invasiveness of cancer cells [15, 17]. In addition, VEGF-C, a member of the VEGF family, modulates angiogenesis and especially lymphangiogenesis which are required for tumor growth and metastasis [18, 19]. The results of Western blot demonstrated in the 5637 cells with HMGN5 knockdown, the level of E-cadherin was significantly enhanced, while the level of VEGF-C was greatly down-regulated when compared with other control cells (Fig. 3). HMGN5 knockdown inhibits the tumor growth in xenograft nude mice To explore the role of HMGN5 in the tumor growth of UBC in vivo, the stable transfectants were used. We established xenograft by subcutaneous injection of the 5637 cells with HMGN5 knockdown or the cells with siRNA control in the armpit area of nude mice (n = 5). After 5 weeks, we found the volume of tumors derived from HMGN5 knockdown cells was significantly smaller than these from siRNA control cells (Fig. 4). The result indicates that HMGN5 knockdown inhibits the tumor growth of 5637 cells in vivo.
Discussion HMGN5, previously known as NBP-45, GARP45 and NSBP1, was eventually identified as a new but typical member of HMGN proteins based on the similar structures and properties as follows: the nucleosome-binding domain (NBD) as the signature of the HMGN family located in the nucleus and negatively charged C terminus [8]. Compared to other members of the HMGN family, the HMGN5 protein has a longer and highly acidic C terminus; additionally, its amount in the cell is estimated to be tenfold lower than that of HMGN1 or HMGN2 [6, 20]. These differences indicate a distinct model of action whereby HMGN5 has a positive role but not negative role like HMGN1 in cancer progression, which has been indentified in varied types of malignant tumors [6, 21, 22]. While the exact mechanism accounts for the role of HMGN5 in tumorigenesis remains unclear, the possible explanation might be that the interaction of HMGN5 with nucleosomes counteracts the compaction of chromatin, then accessibility of DNA is improved with chromatin encompacted and some DNA lesion factors as well as some transcriptional factors take action and thereby modulate the cellular phenotype [4, 23]. To our knowledge, HMGN5 was associated
Fig. 4 Effects of HMGN5 knockdown on tumor growth in vivo. (a) HMGN5 knockdown or siRNA control 5637 cells are subcutaneously injected into the armpit area of nude mice to test their in vivo tumorigenesis ability. Representative nude mice showing the tumors (shown by arrows) derived from HMGN5 knockdown 5637 cells (a) and siRNA control cells (b). The tumors are removed after 5 weeks and shown in picture (c), (d), respectively. (b) The growth curves of transplanted tumors in nude mice (n = 5). Results are expressed as mean ± SD. The results showed that HMGN5 knockdown suppressed the tumor growth in vivo (**p \ 0.01)
with bladder cancer only in one study and more detailed pathogenesis remains to be elucidated [13]. In gene function studies, it is important to knockdown the targeted gene without affecting other genes, RNAi mediated by shRNA is such a specific gene-silencing technology [24]. To further investigate the role of HMGN5 in bladder cancer, here we employed HMGN5 knockdown via RNAi and received a highly inhibitory efficiency at both mRNA and protein level in 5637 cells. After that, we observed the viability of 5637 cells with HMGN5 knockdown significantly decreased by 17 % over 72 h compared to the control, but no increase in apoptosis of 5637 cells was detected, which were similar with the previous study in bladder cancer EJ cell line [13]. However, in the cell cycle analysis, we found that HMGN5 knockdown greatly increased the G1 phrase fraction of 5637 cells and decreased cyclin D1 expression, while a G2/M phase arrest and a decrease in cyclin B1 expression were reported in EJ cells. What’s more, the research in lung cancer cell lines and
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osteosarcoma cell lines even supported a G0/G1 phase arrest after HMGN5 knockdown. These curious results could be explained by the following possibility: previously seen as a general transcription activator, HMGN5 is accepted as a specific transcription modulator now, but HMGN proteins might regulate transcription of distinct genes in distinct circumstances or different types of cells, which leads to the inconsistent results [4, 21, 22, 25]. Furthermore, we also demonstrated that HMGN5 contributed to UBC development in vivo as HMGN5 knockdown suppressed tumor growth of 5637 cells in xenograft nude mice models. In summary, these results suggest that HMGN5 promotes the viability of 5637 cells in vivo and in vitro through increased cell proliferation but not decreased apoptosis. Although the majority of UBCs are stage Ta/T1 tumors, but the patients receiving transurethral resection often suffer greatly from recurrence in suit and the tumors might implant or migrate to other sites of the bladder. Additionally, the progression to muscle-invasive stage is the main clinical concern in these patients, occurring in up to about half of patients [1, 26]. The epithelial–mesenchymal transition (EMT) is a complicated process, in which epithelial cells lose their featured polarity, disassemble cell–cell junctions, and gain typical properties of mesenchymal cells, such as motility and invasiveness [27, 28]. Lots of studies indicate that EMT is tightly correlated with cancer progression in UBC [29]. E-cadherin plays a pivotal role in epithelial cell–cell interactions and is associated with UBC-related EMT intensively. Strong membranous E-cadherin expression throughout normal urothelium is observed, while reduced E-cadherin expression is demonstrated in papillary tumors and even complete absence in advanced invasive tumors [30]. The function of VEGF-C in tumor growth and metastasis has been identified extensively in malignancies, including UBC, for its crucial role in angiogenesis and lymphogenesis [18, 19, 31, 32]. In this study, for the first time, we confirmed HMGN5 knockdown inhibited the invasive potential of 5637 cells in vitro using transwell assay, which was just a speculation in the previous study. Meanwhile, we observed that E-cadherin expression was up-regulated, while the expression of VEGFC was decreased in 5637 cells with HMGN5 knockdown. Since some work found that VEGF-C was often negatively correlated with E-cadherin in the cancerous tissues, the mechanism of VEGF-C participating in metastasis and invasion might be associated with the process of EMT [33– 35]. Therefore, we postulated here aberrant expression of HMGN5 in UBC would accelerate the process of EMT via inhibiting E-cadherin expression and activate abnormal angiogenesis and lymphangiogenesis via inducing expression of VEGF-C and eventually contribute to cancer progression.
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In this study, though we reported some primary findings, some limitations were inevitable. Firstly, all the observations should be verified when HMGN5 is over-expressed to ensure the credibility. Secondly, the mechanism by which HMGN5 affects E-cadherin and VEGF-C is still unknown; more deep and full work is needed. Thirdly, tumor growth and metastasis are complicated courses; more detailed research is needed to confirm our speculation. In our further work, we will pay more attention to cadherin switching and VEGF-C/VEGFR2 or VEGFR3 axis, as well as their correlation with HMGN5 in UBC. In conclusion, here we provide both in vivo and in vitro evidence that HMGN5 contribute to the growth and invasion of UBC 5637 cell line. HMGN5 possibly influences these processes by regulating the expression of cyclin D1, E-cadherin and VEGF-C. The therapy targeting HMGN5 may be a promising approach against UBC development and metastasis. Acknowledgments This work was supported by Grant No. 14JJ3044 from Hunan Provincial Natural Science Foundation of China (website: http://www.hnst.gov.cn/zxgz/zkjj/) and Grant No. B2012-032 from Science Foundation of Health and Family Planning Commission of Hunan Province (website: http://www.hunanwst.gov. cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest interest.
None of the authors have any conflict of
References 1. Burger M, Catto JW, Dalbagni G, Grossman HB, Herr H, Karakiewicz P, et al. Epidemiology and risk factors of urothelial bladder cancer. Eur Urol. 2013;63(2):234–41. doi:10.1016/j.eur uro.2012.07.033. 2. Jacobs BL, Lee CT, Montie JE. Bladder cancer in 2010: how far have we come? CA Cancer J Clin. 2010;60(4):244–72. doi:10. 3322/caac.20077. 3. Kaufman DS, Shipley WU, Feldman AS. Bladder cancer. Lancet. 2009;374(9685):239–49. doi:10.1016/S0140-6736(09)60491-8. 4. Zhu N, Hansen U. Transcriptional regulation by HMGN proteins. Biochim Biophys Acta. 2010;1799(1–2):74–9. doi:10.1016/j. bbagrm.2009.11.006. 5. West KL. HMGN proteins play roles in DNA repair and gene expression in mammalian cells. Biochem Soc Trans. 2004;32(Pt 6):918–9. doi:10.1042/BST0320918. 6. Gerlitz G. HMGNs, DNA repair and cancer. Biochim Biophys Acta. 2010;1799(1–2):80–5. doi:10.1016/j.bbagrm.2009.10.007. 7. Motoyama N, Naka K. DNA damage tumor suppressor genes and genomic instability. Curr Opin Genet Dev. 2004;14(1):11–6. doi:10.1016/j.gde.2003.12.003. 8. Rochman M, Malicet C, Bustin M. HMGN5/NSBP1: a new member of the HMGN protein family that affects chromatin structure and function. Biochim Biophys Acta. 2010;1799(1–2):86–92. doi:10. 1016/j.bbagrm.2009.09.012. 9. Jiang N, Zhou LQ, Zhang XY. Downregulation of the nucleosome-binding protein 1 (NSBP1) gene can inhibit the in vitro and in vivo proliferation of prostate cancer cells. Asian J Androl. 2010;12(5):709–17. doi:10.1038/aja.2010.39.
Med Oncol (2015) 32:136 10. Qu J, Yan R, Chen J, Xu T, Zhou J, Wang M, et al. HMGN5: a potential oncogene in gliomas. J Neurooncol. 2011;104(3):729–36. doi:10.1007/s11060-011-0558-9. 11. Ji SQ, Yao L, Zhang XY, Li XS, Zhou LQ. Knockdown of the nucleosome binding protein 1 inhibits the growth and invasion of clear cell renal cell carcinoma cells in vitro and in vivo. J Exp Clin Cancer Res: CR. 2012;31:22. doi:10.1186/1756-9966-31-22. 12. Green J, Ikram M, Vyas J, Patel N, Proby CM, Ghali L, et al. Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours. Br J Cancer. 2006;94(10):1446–51. doi:10.1038/sj.bjc.6603135. 13. Wahafu W, He ZS, Zhang XY, Zhang CJ, Yao K, Hao H, et al. The nucleosome binding protein NSBP1 is highly expressed in human bladder cancer and promotes the proliferation and invasion of bladder cancer cells. Tumour Biol: J Int Soc Oncodevelopmental Biol Med. 2011;32(5):931–9. doi:10.1007/s13277011-0195-0. 14. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402–8. doi:10.1006/meth. 2001.1262. 15. Mao Q, Li Y, Zheng X, Yang K, Shen H, Qin J, et al. Upregulation of E-cadherin by small activating RNA inhibits cell invasion and migration in 5637 human bladder cancer cells. Biochem Biophys Res Commun. 2008;375(4):566–70. doi:10. 1016/j.bbrc.2008.08.059. 16. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995;9(10):1149–63. 17. Jeanes A, Gottardi CJ, Yap AS. Cadherins and cancer: how does cadherin dysfunction promote tumor progression? Oncogene. 2008;27(55):6920–9. doi:10.1038/onc.2008.343. 18. Benest AV, Harper SJ, Herttuala SY, Alitalo K, Bates DO. VEGF-C induced angiogenesis preferentially occurs at a distance from lymphangiogenesis. Cardiovasc Res. 2008;78(2):315–23. doi:10.1093/cvr/cvm094. 19. Su JL, Yen CJ, Chen PS, Chuang SE, Hong CC, Kuo IH, et al. The role of the VEGF-C/VEGFR-3 axis in cancer progression. Br J Cancer. 2007;96(4):541–5. doi:10.1038/sj.bjc.6603487. 20. Rochman M, Postnikov Y, Correll S, Malicet C, Wincovitch S, Karpova TS, et al. The interaction of NSBP1/HMGN5 with nucleosomes in euchromatin counteracts linker histone-mediated chromatin compaction and modulates transcription. Mol Cell. 2009;35(5):642–56. doi:10.1016/j.molcel.2009.07.002. 21. Zhou X, Yuan B, Yuan W, Wang C, Gao R, Wang J. The expression and clinical significance of high mobility group nucleosome binding domain 5 in human osteosarcoma. Tumour Biol: J Int Soc Oncodevelopmental Biol Med. 2014;35(7): 6539–47. doi:10.1007/s13277-014-1825-0. 22. Weng M, Song F, Chen J, Wu J, Qin J, Jin T, et al. The highmobility group nucleosome-binding domain 5 is highly expressed in breast cancer and promotes the proliferation and invasion of
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23.
24.
25.
26.
27.
28.
29.
30. 31.
32.
33.
34.
35.
breast cancer cells. Tumour Biol J Int Soc Oncodevelopmental Biol Med. 2014. doi:10.1007/s13277-014-2715-1. Pogna EA, Clayton AL, Mahadevan LC. Signalling to chromatin through post-translational modifications of HMGN. Biochim Biophys Acta. 2010;1799(1–2):93–100. doi:10.1016/j.bbagrm.2009.11.018. Lambeth LS, Smith CA. Short hairpin RNA-mediated gene silencing. Methods Mol Biol. 2013;942:205–32. doi:10.1007/9781-62703-119-6_12. Rochman M, Taher L, Kurahashi T, Cherukuri S, Uversky VN, Landsman D, et al. Effects of HMGN variants on the cellular transcription profile. Nucleic Acids Res. 2011;39(10):4076–87. doi:10.1093/nar/gkq1343. van Rhijn BW, Burger M, Lotan Y, Solsona E, Stief CG, Sylvester RJ, et al. Recurrence and progression of disease in non-muscleinvasive bladder cancer: from epidemiology to treatment strategy. Eur Urol. 2009;56(3):430–42. doi:10.1016/j.eururo.2009.06.028. Zavadil J, Haley J, Kalluri R, Muthuswamy SK, Thompson E. Epithelial-mesenchymal transition. Cancer Res. 2008;68(23):9574–7. doi:10.1158/0008-5472.CAN-08-2316. De Wever O, Pauwels P, De Craene B, Sabbah M, Emami S, Redeuilh G, et al. Molecular and pathological signatures of epithelial-mesenchymal transitions at the cancer invasion front. Histochem Cell Biol. 2008;130(3):481–94. doi:10.1007/s00418-008-0464-1. Yun SJ, Kim WJ. Role of the epithelial-mesenchymal transition in bladder cancer: from prognosis to therapeutic target. Korean J Urol. 2013;54(10):645–50. doi:10.4111/kju.2013.54.10.645. Bryan RT, Tselepis C. Cadherin switching and bladder cancer. J Urol. 2010;184(2):423–31. doi:10.1016/j.juro.2010.04.016. Zhang HH, Qi F, Shi YR, Miao JG, Zhou M, He W, et al. RNA interference-mediated vascular endothelial growth factor-C reduction suppresses malignant progression and enhances mitomycin C sensitivity of bladder cancer T24 cells. Cancer Biother radiopharm. 2012;27(5):291–8. doi:10.1089/cbr.2010.0919. Mylona E, Magkou C, Gorantonakis G, Giannopoulou I, Nomikos A, Zarogiannos A, et al. Evaluation of the vascular endothelial growth factor (VEGF)-C role in urothelial carcinomas of the bladder. Anticancer Res. 2006;26(5A):3567–71. Huang KJ, Sui LH. The relevance and role of vascular endothelial growth factor C, matrix metalloproteinase-2 and E-cadherin in epithelial ovarian cancer. Med Oncol. 2012;29(1):318–23. doi:10. 1007/s12032-010-9817-4. Gotte M, Kersting C, Radke I, Kiesel L, Wulfing P. An expression signature of syndecan-1 (CD138), E-cadherin and c-met is associated with factors of angiogenesis and lymphangiogenesis in ductal breast carcinoma in situ. Breast cancer Res: BCR. 2007;9(1):R8. doi:10.1186/bcr1641. Yamashita T, Uramoto H, Onitsuka T, Ono K, Baba T, So T, et al. Association between lymphangiogenesis-/micrometastasisand adhesion-related molecules in resected stage I NSCLC. Lung Cancer. 2010;70(3):320–8. doi:10.1016/j.lungcan.2010.02.013.
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ORIGINAL PAPER
Knockdown of HMGN5 suppresses the viability and invasion of human urothelial bladder cancer 5637 cells in vitro and in vivo Yu Gan1 • Jing Tan1 • Jianfu Yang1 • Yihong Zhou1 • Yingbo Dai1 Leye He1 • Kun Yao1 • Yuxin Tang1
•
Received: 14 March 2015 / Accepted: 16 March 2015 / Published online: 22 March 2015 Ó Springer Science+Business Media New York 2015
Abstract The high-mobility group nucleosome-binding domain 5 (HMGN5) is a new and typical member of HMGN protein family. Numerous studies confirmed that HMGN5 was highly expressed in several kinds of malignant tumors, but its role in cancer progression of urothelial bladder cancer (UBC) has not been fully clarified. This study aimed to further investigate the oncogenic role of HMGN5 in UBC 5637 cells employing in vitro and in vivo models and explored the mechanism. RNA interference was used to down-regulate HMGN5 expression in 5637 cells by a shRNA expression lentiviral vector. Then cell viability, apoptosis and cell cycle distribution, invasion were detected by MTT assay, flow cytometry and transwell assay, respectively. Tumor growth was also evaluated in nude mice. As a result, successful transfection was confirmed using fluorescence microscopy and HMGN5 was efficiently inhibited. HMGN5 knockdown suppressed invasion induced G1/S cell cycle arrest but not apoptosis and thus contributed to decreased cell viability in UBC 5637 cells. Consistent with the cell cycle arrest, the protein expression levels of cyclin D1 were decreased. In vivo study further showed that HMGN5 knockdown affected the tumorigenesis of 5637 cells in nude mice. Western blot also demonstrated that the expression of E-cadherin and VEGFC was decreased in 5637 cells depleted of HMGN5. In conclusion, we provide both in vivo and in vitro evidence that HMGN5 contribute to the growth and invasion of UBC
& Kun Yao [email protected] 1
Department of Urology, The Third Xiangya Hospital of Central South University, 138 Tongzipo Road, Changsha, Hunan 410013, People’s Republic of China
5637 cell line and HMGN5 could be exploited as a target for therapy in UBC. Keywords HMGN5 Urothelial bladder cancer Cell viability Invasion RNA interference
Introduction Urothelial bladder cancer (UBC) is the seventh most common cancer in men and ranks the 17th in women worldwide, with an estimated 110,500 male and 7000 female cases newly diagnosed in the European Union each year [1]. Although nearly 75 % of firstly diagnosed UBCs are noninvasive, it is still a real challenge for urologists to treat them, due to the propensity for disease recurrence and progression [2]. Moreover, radical cystectomy, chemotherapy and radiotherapy for muscle-invasive or metastatic UBCs always have a significantly negative impact on the quality of life and barely bring satisfactory prognosis [3]. So it is important to further understand the pathogenesis of UBC so as to develop effective methods for the early diagnosis and treatment of UBC. High-mobility group N (HMGN) family was previously considered as chromatin architectural proteins only working on regulating the chromatin structure. But latest research indicates that these proteins may have a crucial role in repair of DNA lesions and transcriptional control [4, 5]. Since accumulated DNA lesions which possibly result in genetic mutations and chromosomal aberrations, and aberrant transcriptional level of cancer-related genes have been proved as causes of malignant tumors, it is reasonable to link HMGNs with tumorigenesis [6, 7].
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The high-mobility group nucleosome-binding domain 5 (HMGN5) is a novel and featured member of HMGN protein family with ubiquitous expression in mouse and human tissues [8]. Early studies showed up-regulation of the HMGN5 protein was indentified in certain kinds of malignant tumors, such as prostate cancer, clear cell renal cell carcinoma, squamous cell carcinoma and golima, indicating the carcinogenic role of this protein [9–12]. Recently, Wahafu et al. [13] reported that the HMGN5 protein was over-expressed in human bladder cancer cell lines and tumor tissues, and HMGN5 expression was correlated with the tumor grade and pathologic stage. Though knockdown of HMGN5 via transient transfection was proved to reduce the viability of EJ cells through inducing G2 phase arrest in their study, the contribution of HMGN5 to the invasion ability was speculated. What’s more, further molecular mechanism and in vivo study are also critical to elucidate the role of HMGN5 in cancer progression of UBC. Therefore, in the present study, we investigated the effects of lentivirus mediated HMGN5 knockdown on the growth and invasion of UBC 5637 cells and in vivo tumor growth in nude mice. Our results demonstrated that HMGN5 knockdown induced G1 phase arrest but not apoptosis and thereby reduced the viability of 5637 cells; additionally, the invasion potential was inhibited via upregulating VEGF-C and down-regulating E-cadherin, and tumor growth in vivo was also suppressed.
Methods Cell culture Human UBC 5637 cell line was procured from Yingrun Biotechnologies (Changsha, China). All cells were cultured in RPIM-1640 medium (InvitrogenTM Life Technologies, NY, USA) supplemented with 10 % fetal bovine serum (Invitrogen) under the condition of 37 °C, 5 % CO2 in a standard humidified incubator. Cells in the logarithmic phase were used for the experiments. Lentivirus RNAi construct and transfection A 21-bp fragment within human HMGN5 cDNA (NM_030763) was selected as the target for HMGN5 siRNA, as follows: 50 -GTTGTTGAAGAAGACTACAAT-30 . The other fragment (50 -GACTTCATAAGGCGCATGC-30 ) had no significant homology to human gene sequences and was chosen as siRNA control. Short hairpin RNA (shRNA) fragments were hybridized with synthesized sense and antisense oligonucleotides. The shRNA fragments were then cloned to pYr-Lvsh to construct the lentiviral vectors. The
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positive clones were verified by restriction mapping and direct DNA sequencing. Then the recombinant lentivirus was packaged and concentrated by Yingrun Biotechnologies, and the final virus titer was determined. Four hours before transfection, 5637 cells were placed in serum-free media. Then the cells were infected with the recombinant lentivirus at a multiplicity of infection (MOI) of 50 according to the manufacturer’s protocol. And 1 lg/ ml puromycin (Sigma-Aldrich, St. Louis, MO, USA) could be used for screening positively stable transfectants if needed. Quantitative real-time PCR assay Total RNA was extracted from the cells with Trizol reagent (Invitrogen) according to the manufacturer’s instruction at 72 h post-transfection. The concentration and purity of the RNA samples were determined at 260 nm. First-strand cDNA was generated through reverse transcription via RevertAidTM Reverse Transcriptase (Thermo ScientificTM Life Technologies, NY, USA). The specific primers were synthesized by Sangon (Shanghai, China) and as follows: HMGN5, 50 -CAGGTCAAGGTGATATGAG GCA-30 (forward) and 50 -GCTTGGGCACTTGTATCTATGT-30 (reverse); GAPDH, 50 -TGCACCACCAACTGCTTAGC-30 (forward) and 50 -GGCATGGACTGTGGTCATGA G-30 (reverse). THUNDERBIRDTM SYBRÒ qPCR Mix (Toyobo, Osaka, Japan) was used for real-time PCR, and the reaction system was 20 ll. Thermal cycling was started with a denaturation step for 5 min, followed by 43 cycles done in three steps: 30 s at 95 °C, 30 s at 58 °C and 20 s at 72 °C. GAPDH was used as the internal control, and gene expression was relatively quantified using 2-DDCT method [14]. Western blotting The cells were harvested for the analysis of protein expression at 72 h post-transfection and lysed on ice by RIPA lysis buffer. Protein concentration of lysate was determined by the bicinchoninic acid assay (Beyotime Biotechnology, Shanghai, China). Then protein samples (40 lg) were separated by SDS-PAGE and subsequently transferred onto a PVDF membrane. After blocked with nonfat milk in TBST, the membrane was incubated with primary antibodies against HMGN5 (Abcam, MA, USA), cyclin D1 (Santa Cruz, CA, USA), VEGF-C, E-cadherin and b-actin (Cell Signaling Technology, MA, USA) and then incubated with HRP-conjugated secondary antibody (Santa Cruz). Immunoreactivity signals were developed by ECL reagent (GE Healthcare Bioscience, NJ, USA) and recorded by a fluorescence detection system.
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Cell viability assay The cells were seeded in 96-well plates at a density of 1 9 103/well in a volume of 100 ll and grown overnight. Four hours before transfection, the cells were changed to serum-free medium and transfected with lentivirus. After incubated for 48, 72 or 96 h, 20 ll MTT (5 mg/ml) was added to each well ,then cell plates were put in the incubator for 4 h, and next 150 ll DMSO was added to each well to lyse the cells. Absorbance was measured at 490 nm. Cell cycle analysis The cells were seeded in 25-cm2 flasks. After growing to sufficient confluence, the cells were changed to serum-free medium for 24 h and transfected with lentivirus. The cells were cultured for 72 h and then harvested by gentle trypsinization, extensively washed with cold PBS and collected by centrifugation. Next the cells were resuspended in 0.5 ml PBS and fixed in 1 ml 75 % cold ethanol at 4 °C overnight. After brief centrifugation, the cells were washed once with PBS and incubated in 37 °C for 1 h in 1 ml PBS with 5 ll RNase (10 mg/ml) and then filtered and incubated in the dark for 30 min with 100 lg/ml of propidium iodide. For each tube, 2 9 104 cells were immediately analyzed using a FACS Canto II instrument (BD, CA, USA) and further analyzed using the program with it.
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the upper chamber of each transwell insert (pore size 8 lm, Corning, NY, USA), and then they were incubated at 37 °C for 5 h to make the Matrigel changed from liquid form to gelatum. After hydration of the Matrigel with serum-free medium, 100 ll cells with a density of 5 9 104/ml were seeded in upper chamber; meanwhile, 600 ll medium containing 10 % FBS was filled into the lower chamber. The cells were then cultured at 37 °C for 24 h to allow cells migration. In the end, the noninvasive cells in the upper chamber were gently wiped out with a cotton swab, and the invasive cells attached to the lower chamber were fixed with 4 % paraformaldehyde and stained with 0.1 % crystal violet. Five visual fields were selected randomly, and the cells were counted for each insert and photographed under a light microscope at 2009 magnification. Xenograft The present animal experiment was approved by the Animal Ethics Committee of The Third Xiangya Hospital. Female BALB/c-nu mice (4–5 weeks old, weighed 14–16 g) were maintained in a germ-free environment for 1 week in the animal facility. Then HMGN5 knockdown or siRNA control 5637 cells (4 9 106 in 200 ll medium) were injected subcutaneously in the armpit area. Tumor parameters were measured every week, and tumor volume was calculated by length 9 width2 9 0.5. All the mice were killed after 5 weeks.
Cell apoptosis analysis Data processing An Annexin V-FITC Apoptosis Detection Kit (KeyGEN, Nanjing, China) was used following the manufacturer’s instruction. The cells were seeded in the six-well plates and cultured in the incubator for sufficient confluence. Four hours before transfection, the cells were changed to serumfree medium and then transfected with lentivirus. After 72 h of incubation, the cells were gently trypsinized and washed twice with cold PBS. Then the cells were resuspended in 500 ll 1 9 binding buffer as a density of 1 9 106/ml, and then 100 ll cell suspension was collected, stained with 5 ll of Annexin V-FITC (25 lg/ml) and 5 ll propidium iodide and then incubated on ice in the dark for 15 min. Every sample was diluted with 400 ll PBS and filtered and immediately analyzed using a FACS Canto II instrument (BD).
All the experiments were independently repeated in triplicate, and data were collected. Data were expressed as mean ± standard deviation (mean ± SD) and analyzed for statistical significance using GraphPad Prism 5.01 software (GraphPad Software, CA, USA). Differences among the means of multiple groups were compared based on ANOVA, followed by a Newman–Keuls multiple comparison test. T test was used to compare the difference between two groups. A value of p \ 0.05 was considered as statistically significant.
Results
Cell invasion assay
HMGN5 knockdown decreases the viability of 5637 cells
The invasion capacity of the cells was measured using transwell chamber assay at 72 h post-transfection. The Matrigel (BD) was put into a 4 °C refrigerator overnight for melting. The next day, the Matrigel was diluted by 1:5 with serum-free medium, and 100 ll Matrigel was added to
A total of 5637 cells are derived from a grade II UBC patient, and this cell line is a suitable transfection host [15]. Previous study already identified HMGN5 expression in 5637 cells was higher than normal human urinary tract epithelial cells [13]. Hence, to further investigate the role of HMGN5 in the
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cell biology of UBC cells, we employed 5637 cell line as a model and the loss of function method. A total of 5637 cells were transfected with HMGN5 siRNA or scramble siRNA as RNAi control. Successful transfection of 5637 cells was determined by detecting GFP expression using fluorescence microscopy (Fig. 1). The results showed that lentivirus short hairpin constructs against HMGN5 were efficient and specific in the knockdown of the gene in 5637 cells, with the inhibitory efficiency at mRNA level was 72 ± 5 % based on RT-PCR and at protein level was 60 ± 2 % based on Western blot (Fig. 1). Fig. 1 (a) Confirm the successful transfection by detecting GFP expression using fluorescence microscopy. The transfection efficiency is above 80 % for both two transfection groups (magnification, 9200). (b) Knockdown of HMGN5 in 5637 cells. A total of 5637 cells are transfected with HMGN5 siRNA or scrambled siRNA for 72 h. Quantitative real-time PCR confirmed the HMGN5 mRNA level in the 5637 cells with HMGN5 knockdown is significantly decreased (GAPDH served as the internal control). (c) Similar results are also observed in the protein level, which is confirmed by Western blot analysis (b-actin served as the internal control). Results are expressed as mean ± SD, n = 3. **p \ 0.01 compared to the 5637 cells with scrambled siRNA
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Firstly, cell viability was evaluated by MTT assay and the results showed that HMGN5 knockdown significantly suppressed the viability of 5673 cells over 72 h period (Fig. 2). To ascertain whether this is due to the cell cycle arrest or increased apoptosis, we then performed flow cytometry analysis, and we observed no significant increase in apoptosis in 5637 cells transfected with HMGN5 siRNA (Fig. 2), which was similar to previous report from Wahafu et al. [13]. However, in the cell cycle analysis, we identified an abundant increase in G1 phase fraction of the cells with HMGN5 knockdown, which indicated the G1 phase
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Fig. 2 (a) Detection of cell viability by MTT assay. Cell viability is expressed as the absorbance values. The viability of the 5637 cells with HMGN5 knockdown is suppressed over 72 h after transfection. Results are expressed as mean ± SD, n = 3, *p \ 0.05, **p \ 0.01 compared to the RNAi control 5637 cells. (b) Detection of cell apoptosis by flow cytometry. Statistical analysis of percentage of apoptotic cells failed to show significantly different at 72 h posttransfection among the three groups (p [ 0.05). HMGN5 knockdown induced G1/S phase arrest in 5637 cells at 72 h post-transfection.
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(c) a Cell cycle distribution is performed by flow cytometry. The percentage of cells at G1 phase is moderately increased in the 5367 cells with HMGN5 knockdown compared to that in the 5637 cells with scrambled siRNA (p \ 0.01). b Cyclin D1 is a sensitive checkpoint of G1 phase in cell cycle. Western blot analysis confirmed the expression of cyclin D1 in the 5367 cells with HMGN5 knockdown is moderately suppressed (p \ 0.05). b-Actin served as the internal control. Results are expressed as mean ± SD, n = 3, **p \ 0.01
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arrest in these cells (Fig. 2). Cyclin D1 is one of the crucial checkpoints of G1/S transition [16]. To further confirm HMGN5 modulates cell cycle progression of 5637 cells, we examined the level of cyclin D1 by Western blot analysis and found it was also significantly decreased in the cells with HMGN5 knockdown (Fig. 2). Collectively, these results suggest that HMGN5 might modulate the G1 phase of cell cycle but not apoptosis and contribute to the viability of 5637 cells.
Fig. 3 (a) Test of cell invasiveness by transwell assay. The cells penetrating the filter membrane at 72 h posttransfection are stained with crystal violet and are shown as blue under a light microscope (magnification, 9 200). a Blank control. b RNAi control. c HMGN5-RNAi. d The quantitative results showed a greatly reduced invasiveness of the 5637 cells with HMGN5 knockdown compared to that of RNAi control cells. (b) Western blot for HMGN5, E-cadherin, VEGF-C and b-actin at 72 h post-transfection. HMGN5 knockdown inhibited the expression of E-cadherin (p \ 0.01) and VEGF-C (p \ 0.01) in 5637 cells. bActin served as the internal control
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HMGN5 knockdown inhibits the invasion of 5637 cells Next we utilize transwell invasion assay to confirm the previous speculation in the role of HMGN5 in UBC cells invasion. The filter was stained with crystal violet and inspected under the microscope (Fig. 3); we found HMGN5 siRNA significantly inhibited the invasiveness of 5637 cells. The number of cells penetrating the Matrigel was
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20.7 ± 4.2 in HMGN5 siRNA group versus 65.4 ± 24.5 in blank control group (p \ 0.01; Fig. 3). E-cadherin, a calcium-dependent transmembrane glycoprotein, is required for cell–cell adhesion and viewed as a key tumor suppressor related with invasiveness of cancer cells [15, 17]. In addition, VEGF-C, a member of the VEGF family, modulates angiogenesis and especially lymphangiogenesis which are required for tumor growth and metastasis [18, 19]. The results of Western blot demonstrated in the 5637 cells with HMGN5 knockdown, the level of E-cadherin was significantly enhanced, while the level of VEGF-C was greatly down-regulated when compared with other control cells (Fig. 3). HMGN5 knockdown inhibits the tumor growth in xenograft nude mice To explore the role of HMGN5 in the tumor growth of UBC in vivo, the stable transfectants were used. We established xenograft by subcutaneous injection of the 5637 cells with HMGN5 knockdown or the cells with siRNA control in the armpit area of nude mice (n = 5). After 5 weeks, we found the volume of tumors derived from HMGN5 knockdown cells was significantly smaller than these from siRNA control cells (Fig. 4). The result indicates that HMGN5 knockdown inhibits the tumor growth of 5637 cells in vivo.
Discussion HMGN5, previously known as NBP-45, GARP45 and NSBP1, was eventually identified as a new but typical member of HMGN proteins based on the similar structures and properties as follows: the nucleosome-binding domain (NBD) as the signature of the HMGN family located in the nucleus and negatively charged C terminus [8]. Compared to other members of the HMGN family, the HMGN5 protein has a longer and highly acidic C terminus; additionally, its amount in the cell is estimated to be tenfold lower than that of HMGN1 or HMGN2 [6, 20]. These differences indicate a distinct model of action whereby HMGN5 has a positive role but not negative role like HMGN1 in cancer progression, which has been indentified in varied types of malignant tumors [6, 21, 22]. While the exact mechanism accounts for the role of HMGN5 in tumorigenesis remains unclear, the possible explanation might be that the interaction of HMGN5 with nucleosomes counteracts the compaction of chromatin, then accessibility of DNA is improved with chromatin encompacted and some DNA lesion factors as well as some transcriptional factors take action and thereby modulate the cellular phenotype [4, 23]. To our knowledge, HMGN5 was associated
Fig. 4 Effects of HMGN5 knockdown on tumor growth in vivo. (a) HMGN5 knockdown or siRNA control 5637 cells are subcutaneously injected into the armpit area of nude mice to test their in vivo tumorigenesis ability. Representative nude mice showing the tumors (shown by arrows) derived from HMGN5 knockdown 5637 cells (a) and siRNA control cells (b). The tumors are removed after 5 weeks and shown in picture (c), (d), respectively. (b) The growth curves of transplanted tumors in nude mice (n = 5). Results are expressed as mean ± SD. The results showed that HMGN5 knockdown suppressed the tumor growth in vivo (**p \ 0.01)
with bladder cancer only in one study and more detailed pathogenesis remains to be elucidated [13]. In gene function studies, it is important to knockdown the targeted gene without affecting other genes, RNAi mediated by shRNA is such a specific gene-silencing technology [24]. To further investigate the role of HMGN5 in bladder cancer, here we employed HMGN5 knockdown via RNAi and received a highly inhibitory efficiency at both mRNA and protein level in 5637 cells. After that, we observed the viability of 5637 cells with HMGN5 knockdown significantly decreased by 17 % over 72 h compared to the control, but no increase in apoptosis of 5637 cells was detected, which were similar with the previous study in bladder cancer EJ cell line [13]. However, in the cell cycle analysis, we found that HMGN5 knockdown greatly increased the G1 phrase fraction of 5637 cells and decreased cyclin D1 expression, while a G2/M phase arrest and a decrease in cyclin B1 expression were reported in EJ cells. What’s more, the research in lung cancer cell lines and
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osteosarcoma cell lines even supported a G0/G1 phase arrest after HMGN5 knockdown. These curious results could be explained by the following possibility: previously seen as a general transcription activator, HMGN5 is accepted as a specific transcription modulator now, but HMGN proteins might regulate transcription of distinct genes in distinct circumstances or different types of cells, which leads to the inconsistent results [4, 21, 22, 25]. Furthermore, we also demonstrated that HMGN5 contributed to UBC development in vivo as HMGN5 knockdown suppressed tumor growth of 5637 cells in xenograft nude mice models. In summary, these results suggest that HMGN5 promotes the viability of 5637 cells in vivo and in vitro through increased cell proliferation but not decreased apoptosis. Although the majority of UBCs are stage Ta/T1 tumors, but the patients receiving transurethral resection often suffer greatly from recurrence in suit and the tumors might implant or migrate to other sites of the bladder. Additionally, the progression to muscle-invasive stage is the main clinical concern in these patients, occurring in up to about half of patients [1, 26]. The epithelial–mesenchymal transition (EMT) is a complicated process, in which epithelial cells lose their featured polarity, disassemble cell–cell junctions, and gain typical properties of mesenchymal cells, such as motility and invasiveness [27, 28]. Lots of studies indicate that EMT is tightly correlated with cancer progression in UBC [29]. E-cadherin plays a pivotal role in epithelial cell–cell interactions and is associated with UBC-related EMT intensively. Strong membranous E-cadherin expression throughout normal urothelium is observed, while reduced E-cadherin expression is demonstrated in papillary tumors and even complete absence in advanced invasive tumors [30]. The function of VEGF-C in tumor growth and metastasis has been identified extensively in malignancies, including UBC, for its crucial role in angiogenesis and lymphogenesis [18, 19, 31, 32]. In this study, for the first time, we confirmed HMGN5 knockdown inhibited the invasive potential of 5637 cells in vitro using transwell assay, which was just a speculation in the previous study. Meanwhile, we observed that E-cadherin expression was up-regulated, while the expression of VEGFC was decreased in 5637 cells with HMGN5 knockdown. Since some work found that VEGF-C was often negatively correlated with E-cadherin in the cancerous tissues, the mechanism of VEGF-C participating in metastasis and invasion might be associated with the process of EMT [33– 35]. Therefore, we postulated here aberrant expression of HMGN5 in UBC would accelerate the process of EMT via inhibiting E-cadherin expression and activate abnormal angiogenesis and lymphangiogenesis via inducing expression of VEGF-C and eventually contribute to cancer progression.
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In this study, though we reported some primary findings, some limitations were inevitable. Firstly, all the observations should be verified when HMGN5 is over-expressed to ensure the credibility. Secondly, the mechanism by which HMGN5 affects E-cadherin and VEGF-C is still unknown; more deep and full work is needed. Thirdly, tumor growth and metastasis are complicated courses; more detailed research is needed to confirm our speculation. In our further work, we will pay more attention to cadherin switching and VEGF-C/VEGFR2 or VEGFR3 axis, as well as their correlation with HMGN5 in UBC. In conclusion, here we provide both in vivo and in vitro evidence that HMGN5 contribute to the growth and invasion of UBC 5637 cell line. HMGN5 possibly influences these processes by regulating the expression of cyclin D1, E-cadherin and VEGF-C. The therapy targeting HMGN5 may be a promising approach against UBC development and metastasis. Acknowledgments This work was supported by Grant No. 14JJ3044 from Hunan Provincial Natural Science Foundation of China (website: http://www.hnst.gov.cn/zxgz/zkjj/) and Grant No. B2012-032 from Science Foundation of Health and Family Planning Commission of Hunan Province (website: http://www.hunanwst.gov. cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Conflict of interest interest.
None of the authors have any conflict of
References 1. Burger M, Catto JW, Dalbagni G, Grossman HB, Herr H, Karakiewicz P, et al. Epidemiology and risk factors of urothelial bladder cancer. Eur Urol. 2013;63(2):234–41. doi:10.1016/j.eur uro.2012.07.033. 2. Jacobs BL, Lee CT, Montie JE. Bladder cancer in 2010: how far have we come? CA Cancer J Clin. 2010;60(4):244–72. doi:10. 3322/caac.20077. 3. Kaufman DS, Shipley WU, Feldman AS. Bladder cancer. Lancet. 2009;374(9685):239–49. doi:10.1016/S0140-6736(09)60491-8. 4. Zhu N, Hansen U. Transcriptional regulation by HMGN proteins. Biochim Biophys Acta. 2010;1799(1–2):74–9. doi:10.1016/j. bbagrm.2009.11.006. 5. West KL. HMGN proteins play roles in DNA repair and gene expression in mammalian cells. Biochem Soc Trans. 2004;32(Pt 6):918–9. doi:10.1042/BST0320918. 6. Gerlitz G. HMGNs, DNA repair and cancer. Biochim Biophys Acta. 2010;1799(1–2):80–5. doi:10.1016/j.bbagrm.2009.10.007. 7. Motoyama N, Naka K. DNA damage tumor suppressor genes and genomic instability. Curr Opin Genet Dev. 2004;14(1):11–6. doi:10.1016/j.gde.2003.12.003. 8. Rochman M, Malicet C, Bustin M. HMGN5/NSBP1: a new member of the HMGN protein family that affects chromatin structure and function. Biochim Biophys Acta. 2010;1799(1–2):86–92. doi:10. 1016/j.bbagrm.2009.09.012. 9. Jiang N, Zhou LQ, Zhang XY. Downregulation of the nucleosome-binding protein 1 (NSBP1) gene can inhibit the in vitro and in vivo proliferation of prostate cancer cells. Asian J Androl. 2010;12(5):709–17. doi:10.1038/aja.2010.39.
Med Oncol (2015) 32:136 10. Qu J, Yan R, Chen J, Xu T, Zhou J, Wang M, et al. HMGN5: a potential oncogene in gliomas. J Neurooncol. 2011;104(3):729–36. doi:10.1007/s11060-011-0558-9. 11. Ji SQ, Yao L, Zhang XY, Li XS, Zhou LQ. Knockdown of the nucleosome binding protein 1 inhibits the growth and invasion of clear cell renal cell carcinoma cells in vitro and in vivo. J Exp Clin Cancer Res: CR. 2012;31:22. doi:10.1186/1756-9966-31-22. 12. Green J, Ikram M, Vyas J, Patel N, Proby CM, Ghali L, et al. Overexpression of the Axl tyrosine kinase receptor in cutaneous SCC-derived cell lines and tumours. Br J Cancer. 2006;94(10):1446–51. doi:10.1038/sj.bjc.6603135. 13. Wahafu W, He ZS, Zhang XY, Zhang CJ, Yao K, Hao H, et al. The nucleosome binding protein NSBP1 is highly expressed in human bladder cancer and promotes the proliferation and invasion of bladder cancer cells. Tumour Biol: J Int Soc Oncodevelopmental Biol Med. 2011;32(5):931–9. doi:10.1007/s13277011-0195-0. 14. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402–8. doi:10.1006/meth. 2001.1262. 15. Mao Q, Li Y, Zheng X, Yang K, Shen H, Qin J, et al. Upregulation of E-cadherin by small activating RNA inhibits cell invasion and migration in 5637 human bladder cancer cells. Biochem Biophys Res Commun. 2008;375(4):566–70. doi:10. 1016/j.bbrc.2008.08.059. 16. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995;9(10):1149–63. 17. Jeanes A, Gottardi CJ, Yap AS. Cadherins and cancer: how does cadherin dysfunction promote tumor progression? Oncogene. 2008;27(55):6920–9. doi:10.1038/onc.2008.343. 18. Benest AV, Harper SJ, Herttuala SY, Alitalo K, Bates DO. VEGF-C induced angiogenesis preferentially occurs at a distance from lymphangiogenesis. Cardiovasc Res. 2008;78(2):315–23. doi:10.1093/cvr/cvm094. 19. Su JL, Yen CJ, Chen PS, Chuang SE, Hong CC, Kuo IH, et al. The role of the VEGF-C/VEGFR-3 axis in cancer progression. Br J Cancer. 2007;96(4):541–5. doi:10.1038/sj.bjc.6603487. 20. Rochman M, Postnikov Y, Correll S, Malicet C, Wincovitch S, Karpova TS, et al. The interaction of NSBP1/HMGN5 with nucleosomes in euchromatin counteracts linker histone-mediated chromatin compaction and modulates transcription. Mol Cell. 2009;35(5):642–56. doi:10.1016/j.molcel.2009.07.002. 21. Zhou X, Yuan B, Yuan W, Wang C, Gao R, Wang J. The expression and clinical significance of high mobility group nucleosome binding domain 5 in human osteosarcoma. Tumour Biol: J Int Soc Oncodevelopmental Biol Med. 2014;35(7): 6539–47. doi:10.1007/s13277-014-1825-0. 22. Weng M, Song F, Chen J, Wu J, Qin J, Jin T, et al. The highmobility group nucleosome-binding domain 5 is highly expressed in breast cancer and promotes the proliferation and invasion of
Page 9 of 9 136
23.
24.
25.
26.
27.
28.
29.
30. 31.
32.
33.
34.
35.
breast cancer cells. Tumour Biol J Int Soc Oncodevelopmental Biol Med. 2014. doi:10.1007/s13277-014-2715-1. Pogna EA, Clayton AL, Mahadevan LC. Signalling to chromatin through post-translational modifications of HMGN. Biochim Biophys Acta. 2010;1799(1–2):93–100. doi:10.1016/j.bbagrm.2009.11.018. Lambeth LS, Smith CA. Short hairpin RNA-mediated gene silencing. Methods Mol Biol. 2013;942:205–32. doi:10.1007/9781-62703-119-6_12. Rochman M, Taher L, Kurahashi T, Cherukuri S, Uversky VN, Landsman D, et al. Effects of HMGN variants on the cellular transcription profile. Nucleic Acids Res. 2011;39(10):4076–87. doi:10.1093/nar/gkq1343. van Rhijn BW, Burger M, Lotan Y, Solsona E, Stief CG, Sylvester RJ, et al. Recurrence and progression of disease in non-muscleinvasive bladder cancer: from epidemiology to treatment strategy. Eur Urol. 2009;56(3):430–42. doi:10.1016/j.eururo.2009.06.028. Zavadil J, Haley J, Kalluri R, Muthuswamy SK, Thompson E. Epithelial-mesenchymal transition. Cancer Res. 2008;68(23):9574–7. doi:10.1158/0008-5472.CAN-08-2316. De Wever O, Pauwels P, De Craene B, Sabbah M, Emami S, Redeuilh G, et al. Molecular and pathological signatures of epithelial-mesenchymal transitions at the cancer invasion front. Histochem Cell Biol. 2008;130(3):481–94. doi:10.1007/s00418-008-0464-1. Yun SJ, Kim WJ. Role of the epithelial-mesenchymal transition in bladder cancer: from prognosis to therapeutic target. Korean J Urol. 2013;54(10):645–50. doi:10.4111/kju.2013.54.10.645. Bryan RT, Tselepis C. Cadherin switching and bladder cancer. J Urol. 2010;184(2):423–31. doi:10.1016/j.juro.2010.04.016. Zhang HH, Qi F, Shi YR, Miao JG, Zhou M, He W, et al. RNA interference-mediated vascular endothelial growth factor-C reduction suppresses malignant progression and enhances mitomycin C sensitivity of bladder cancer T24 cells. Cancer Biother radiopharm. 2012;27(5):291–8. doi:10.1089/cbr.2010.0919. Mylona E, Magkou C, Gorantonakis G, Giannopoulou I, Nomikos A, Zarogiannos A, et al. Evaluation of the vascular endothelial growth factor (VEGF)-C role in urothelial carcinomas of the bladder. Anticancer Res. 2006;26(5A):3567–71. Huang KJ, Sui LH. The relevance and role of vascular endothelial growth factor C, matrix metalloproteinase-2 and E-cadherin in epithelial ovarian cancer. Med Oncol. 2012;29(1):318–23. doi:10. 1007/s12032-010-9817-4. Gotte M, Kersting C, Radke I, Kiesel L, Wulfing P. An expression signature of syndecan-1 (CD138), E-cadherin and c-met is associated with factors of angiogenesis and lymphangiogenesis in ductal breast carcinoma in situ. Breast cancer Res: BCR. 2007;9(1):R8. doi:10.1186/bcr1641. Yamashita T, Uramoto H, Onitsuka T, Ono K, Baba T, So T, et al. Association between lymphangiogenesis-/micrometastasisand adhesion-related molecules in resected stage I NSCLC. Lung Cancer. 2010;70(3):320–8. doi:10.1016/j.lungcan.2010.02.013.
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