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1、精品论文Aberrant Expression of ZNF268 Alters the Growth andMigration of Ovarian Cancer Cells5HU Li1, WANG Wei1, CAI Jinyang1, LUO Jun2, HUANG Yi3, XIONG Shilu3, LI Wenxin1, GUO Mingxiong1(1. College of Life Sciences, Wuhan University;2. Department of Pathology, Zhongnan Hospital, Wuhan University;3. Dep
2、artment of Gynecologic Oncology, Hubei Cancer Hospital)10Abstract: Ovarian cancer is one of the most lethal gynaecologic cancers worldwide. However, the mechanism underlying ovarian carcinogenesis is not well understood. Here, we show that ZNF268 isoverexpressed in human ovarian carcinomas. ZNF268 k
3、nockdown increased the viability, colonyformation, and the growth of in vivo xenografts of ovarian carcinoma SKOV-3 cells, whereas SKOV-3 cell migration was inhibited by ZNF268 knockdown. Furthermore, we demonstrated that ZNF26815knockdown may increase SKOV-3 cell growth by promoting cell cycle prog
4、ression. Our findings suggest that ZNF268 is a novel protein involved in ovarian carcinogenesis and may help us betterunderstand the mechanism of ovarian carcinogenesis.Key words: ZNF268; Ovarian cancer; SKOV-3 cells200IntroductionKrppel-associated box (KRAB)-containing zinc finger (KRAB-ZNF) protei
5、ns, which represent the largest single family of transcriptional regulators in mammals 1, have been shown to regulate gene expression by binding to target DNA sequences through the zinc finger domain, thereby allowing KRAB to repress transcription 2,3. Nevertheless, little is known about the25biolog
6、ical functions of KRAB-ZNF proteins 4. ZNF268, which was isolated from a humanembryo cDNA library 5, is a typical KRAB-containing zinc finger protein that has been found to produce eight splice variants and be translated into two proteins: ZNF268a and ZNF268b2 6. ZNF268a contains a KRAB domain and a
7、s many as 24 zinc fingers and may function as a transcriptional repressor 7, while ZNF268b2 consists of only the zinc finger domain and30contributes to human cervical cancer via NF-B signalling pathway 8. The ZNF268 promoter islocated in the first exon of the gene and can be regulated by cAMP respon
8、se element binding protein 2 (CREB-2) 9. Previous studies suggest that ZNF268 may be involved in human foetal liver development 10, haematological diseases 11,12,13 and cervical cancer development 8. Based on recent tissue microarray results, ZNF268 may be a multifunctional molecule that can functio
9、n35as either a promoter or a suppressor, depending on the cancer subtype 8. This hypothesis issupported by recent findings that indicate that ZNF268 knockdown promotes the proliferation of erythroleukemia K562 cells 14, while inhibiting the growth of cervical cancer HeLa cells 8. However, the functi
10、on of ZNF268 in ovarian tissues remains to be determined.Ovarian cancer is one of the most lethal gynaecologic cancers and the seventh leading cause40of cancer death among women worldwide 15. Its incidence in Asian countries is increasing 16.The high mortality associated with ovarian cancer is large
11、ly due to the asymptomatic nature of early stages of the disease (prior to the development of widespread metastases) and the significantFoundations: This work was supported by National High Technology Research and Development Program of China (863 Program) Grant (2006AA02A306), National Natural Scie
12、nce Foundation of China Grants (30871245 and 31271511), and a Specialized Research Fund for the Doctoral Program of Higher Education of China Grant (200804861004).Brief author introduction:胡丽（1988-），女，硕士研究生，主要研究方向为肿瘤生物学Correspondance author: 郭明雄(1971-),男,博士,副教授,硕士研究生导师,主要研究方向为发育生物学和肿瘤 生物学. E-mail: -
13、 10 -failure rate of chemotherapy for curing advanced disease 17,18. To date, the pathogenic mechanisms underlying the development of human ovarian cancers are too complicated to be fully45understood 19. Therefore, there is an urgent need to investigate the cellular and molecularmechanisms underlyin
14、g ovarian carcinogenesis and to identify novel therapeutic targets that will improve the survival of patients with ovarian cancer.In this study, we demonstrated that ZNF268 was overexpressed in human ovarian cancer tissues and that ZNF268 knockdown increased the proliferation of, while simultaneousl
15、y50decreasing the migration of, SKOV-3 ovarian cancer cells. The effects of ZNF268 on SKOV-3 cell growth may be mediated by altering cell cycle progression. The results obtained from the current study will give us a better understanding of the molecular mechanisms underlying ovarian cancer progressi
16、on and investigate the potential of ZNF268 as a novel therapeutic target for ovarian cancer.551Materials and methods1.1Cell culture, tissue specimens and immunohistochemistrySKOV-3 cells (CCTCC, Wuhan, China) were grown in McCoys 5A medium supplementedwith 10% FBS (Invitrogen), penicillin (100 units
17、/ml), and streptomycin (100 g/ml) at 37C in a5% CO2 incubator. Four paraffin-embedded normal ovarian specimens and 20 paraffin-embedded60ovarian carcinoma specimens were obtained during routine clinical practice from women who were undergoing either biopsy or surgery at the Department of Pathology,
18、Zhongnan Hospital, Wuhan University in China. Our study was approved by the Regional Committee of Medical Research Ethics in China, and informed consent was obtained from each patient.All immunohistochemical staining assays were performed by Jiayuan Quantum Dots65Company (Wuhan, China) according to
19、the standard procedure. The expression levels of the examined proteins were scored by two independent pathologists without knowledge of the clinical or histopathological data. The expression levels were scored as follows: 0 points (-, no detectable staining), 1 point (+, weak staining), 2 points (+,
20、 clear but not strong staining), and 3 points (+,strong staining) 20.701.2Establishment of ZNF268 stable knockdown SKOV-3 cellsSKOV-3 cells were seeded in 60-mm dishes at a density of 3.0 x 104 cells/dish. When the cells had reached approximately 70% confluence, they were infected with shZNF268 or s
21、h control lentiviral particles as described 14. Flow cytometry was used to sort the GFP-positive cells. Reduced ZNF268 expression was confirmed at both the mRNA and protein levels 14.751.3Western blottingCells were collected and lysed in RIPA buffer on ice for 15 min, followed by centrifugation at 1
22、2,000 rpm at 4C for 10 min. The supernatants were collected and subjected to western blotting according to the standard procedure. The antibodies used for western blotting analysis were as follows: the actin antibody was purchased from Santa Cruz80Biotechnology; the cyclin D2, cyclin E2 and CDK2 ant
23、ibodies were purchased from Cell Signaling Technology; the anti-SD antibody for the detection of ZNF268 was used as previously described 6.1.4RNA isolation, reverse transcription and quantitative real-time PCRRNA was extracted using TRIZOL reagent (Invitrogen). cDNA was prepared according to85909510
24、0105110115120125the manufacturers recommended instructions (Toyobo). Real-time PCR was performed and analysed using an ABI 7500 Detection System. The relative mRNA levels of ZNF268 in each sample were normalised to GAPDH as described 14.1.5MTT AssaySKOV-3 cells were plated in 96-well cell culture pl
25、ates at a density of 2.0 x 103 cells/well. At designatedtimepoints,thecellswereincubatedwith20lof3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT, 5 mg/ml) for 2 h, followed by solubilisation for 10 min in DMSO (100 l/well) on a shaker at room temperature. The absorbance was det
26、ermined at 570 nm using a microplate reader (Biotechnology).1.6Soft agar assayCells were trypsinised and suspended in 2 ml of top agar containing 10% FBS and 0.3% agarose. The mixture was then plated onto 60-mm dishes containing 2 ml of bottom agar with 10% FBS and 0.6% agarose. After incubation for
27、 3 weeks, 1 ml of a 1 mg/ml solution of2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride was added. After 4 h, the plates were photographed and the number of colonies was counted.1.7Cell cycle progression and apoptosis assaysCell cycle progression and apoptosis were assessed using a o
28、w cytometric analysis to measure DNA content. Briefly, SKOV-3 cells (1.0 x 106) were collected, washed twice with PBS, and resuspended in ice cold 70% ethanol for 2 h at 4C. The cells were then washed twice with PBS and digested using RNase A (1 mg/ml) at 37C for 30 min. The cells were stained withP
29、ropidium Iodide (PI, 5 g/ml) for 1 h at 4C and analysed using a Beckman Coulter Epics XL Flow Cytometer.1.8Nude mouse tumour formation assayFive-week-old male nude mice (Balb/c nu/nu) were purchased from SJA Lab Animal Limited Company (China). The animals were housed in a specific pathogen-free faci
30、lity and kept in a temperature-controlled environment on a 12-h light/dark cycle with free access to sterilised food and autoclaved water. SKOV-3 cells in the exponential growth phase were trypsinised into single-cell suspensions and injected subcutaneously into nude mice. All mice were maintained f
31、or30 days before they were sacrificed. All animal experiments were approved by the Animal Research Ethics Board of Wuhan University in China and were conducted in compliance with the institutional guidelines for the care of experimental animals.1.9Wound healing migration assayEither SKOV-3 cells or
32、HeLa cells (1 x 105) were plated onto 6-well plates and allowed to create a confluent monolayer. The cell monolayer was then scratched in a straight line to make a “scratch wound” with a 0.2 ml pipette tip, and the cell debris was removed by washing the cells with phosphate-buffered saline. McCoys 5
33、A medium (for SKOV-3 cells) or DMEM medium (for HeLa cells) supplemented with 1% FBS was added, and the closure of the scratch was photographed at 0 h, 24 h, and 48 h.1.10 StatisticsThe data are represented as the mean SD of the samples. All experiments were repeated three times. A two-tailed Studen
34、t t test was used to compare differences between two experimental groups. A P value less than 0.05 was considered to be statistically significant.2Results1301351401451502.1ZNF268 is overexpressed in human ovarian cancer tissuesWe recently observed different expression patterns of ZNF268 in normal hu
35、man ovarian tissues compared to those of ovarian cancer tissues using the tissue microarray method. ZNF268 was overexpressed in most of the ovarian cancer tissues (84%), while ZNF268 expression was rarely detected in the normal tissues 8. To confirm these results, more ovarian tissues (4 normaland 2
36、0 cancerous specimens) were obtained and subjected to immunohistochemistry with an anti-SD antibody to detect ZNF268 expression 6. Most of the cancerous samples displayed high levels of ZNF268 expression (75% with +/+ staining) compared with the expression levels observed in normal tissues (75% with
37、 -/+ staining) (Figure 1A and 1B). These results suggested that ZNF268 was overexpressed in human ovarian cancer tissues.Figure 1. ZNF268 expression in human ovarian tissues. (A) Representative images of ZNF268 immunohistochemical staining in ovarian tissues. Three cases of cancerous ovarian tissues
38、 are shown. Anti-SD antibodies were used to detect ZNF268 expression using immunohistochemistry 6. The scale bars represent 50 m. (B) The accumulated percentage of ZNF268 expression in normal and cancerous ovarian tissues. The number in brackets indicates the number of specimensinvestigated.2.2ZNF26
39、8 knockdown promotes SKOV-3 cell growthTo investigate the biological function of ZNF268 overexpression in human ovarian cancer tissues, ZNF268 knockdown in ovarian cancer SKOV-3 cells (shZNF268) was established using the siRNA method (Figure 2A). The expression of ZNF268 mRNA and the levels of the p
40、roteins (ZNF268a and ZNF268b2) encoded by the ZNF268 gene were decreased in shZNF268 cells (Figure 2A). Next, we analysed the effect of ZNF268 knockdown on SKOV-3 cell growth using the MTT assay. The results showed that the growth rate of shZNF268 cells was increased 2 days after the cells were plat
41、ed, and the greatest increase in growth rate occurred after 5 days (Figure2B). The ability to form colonies in soft agar is considered to be an important characteristic of155160165170175tumour growth in vitro. Therefore, we examined the ability of shZNF268 SKOV-3 cells to form colonies in soft agar.
42、 Consistent with the results of the MTT assay, the number of colonies was significantly increased in the shZNF268 SKOV-3 cells compared to that of the sh control cells (Figure 2C).Figure 2. ZNF268 knockdown enhances SKOV-3 cell growth in both in vitro models and in vivo xenografts. (A) ZNF268 knockd
43、own was confirmed at both the mRNA and protein levels. SKOV-3 cells were infected with lentiviral particles carrying either the ZNF268 hairpin (shZNF268) or control (sh control) sequences and subjected to real-time PCR (left panel) and a western blot (right panel) analysis to examine ZNF268 knockdow
44、n. An anti-SD antibody was used to detect ZNF268a/ZNF268b2 protein levels. (B) ZNF268 knockdown increased SKOV-3 cellviability. SKOV-3 cells were seeded at 3.0 x 103 cells per well in 96-well plates. The growth ratewas determined using an MTT assay at the indicated time points. (C) ZNF268 knockdown
45、increased colony formation in soft agar. Cells were seeded at a density of 4.0 x 103 per 60-mm plate.Threeweekslater,thecellcolonieswerestainedwith2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride for visualisation (upper panel)and statistic analysis (bottom panel). *, P0.05; *, P0.01
46、; *, P0.001. (D) shZNF268SKOV-3 cell xenografts exhibit increased growth. shZNF268 or sh control SKOV-3 cells (5.0 x106) were subcutaneously injected into nude mice. After 40 days, the mice were sacrificed and the tumours were removed, as shown in the bottom panel.To further study the function of ZNF268, we used an in vivo xenograft model. After180185190