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Knockdown of Zinc Finger Protein X-Linked Inhibits Prostate Cancer

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Knockdown of Zinc Finger Protein X-Linked Inhibits Prostate Cancer Cancer Gene Therapy (2012) 19, 684–689 & 2012 Nature America, Inc. All rights reserved 0929-1903/12 www.nature.com/cgt ORIGINAL ARTICLE Knockdown of zinc finger protein X-linked inhibits prostate cancer cell proliferation and induces apoptosis by activating caspase-3 and caspase-9 H Jiang1,4, L Zhang2,4, J Liu2, Z Chen3,RNa2, G Ding2, H Zhang3 and Q Ding1 Zinc finger protein X-linked (ZFX) is a highly conservative mammalian gene with related functions in cell survival and proliferation. However, there are limited reports on regulation of ZFX as a therapeutic target in cancer treatment. In this study, the expression of ZFX in prostate cancer with matched normal adjacent tissues (n ¼ 45) and benign prostatic hyperplasia (BPH) tissues (n ¼ 16) were observed by immunohistochemical analysis. The effect of lentiviral siRNA (small interference RNA)- mediated dysfunction of ZFX on the proliferation of prostate cancer cells was studied. ZFX mRNA and protein expression levels in prostate cancer cells (PC-3 and DU145) were analyzed by western blotting and real-time polymerase chain reaction (RT-PCR). The effects of siRNA targeting ZFX on growth, cell cycle and apoptosis of PC-3 cells were evaluated by MTT assay and flow cytometry. We also investigated the effect of ZFX deletion on the activation of caspase-1, -3 and -9 by western blotting and colorimetric assay. Prostate cancer specimens appeared significantly higher (42.2% of cases being positive) than that observed in normal adjacent tissues (11.8% of cases being positive) and BPH tissues (12.5% of cases being positive). Repression of ZFX in the prostate cancer cells effectively suppressed the cellular proliferation and colony-formation ability, and led to G1 phase cell cycle arrest. Moreover, inhibition of ZFX-induced cell apoptosis by activating caspase-1, -3 and -9. In conclusion, ZFX represents the prominent role in the progression of prostate cancer and may be a promising therapy target for prostate cancer. Cancer Gene Therapy (2012) 19, 684–689; doi:10.1038/cgt.2012.53; published online 17 August 2012 Keywords: prostate cancer; zinc finger protein X-linked; proliferation; apoptosis INTRODUCTION There are a number of studies reporting diverse structures of zinc 6,7 Prostate cancer is one of the most frequently diagnosed non- finger proteins corresponding to novel topologies. Zinc finger cutaneous malignancy and the sixth leading cause of cancer death protein X-linked (ZFX) is a member of the krueppel C2H2-type in males, accounting for 14% (903 500) of the total new cancer zinc-finger protein family and is on the X chromosome. This is cases and 6% (258 400) of the total cancer deaths in males in structurally similar to a related gene on the Y chromosome, zinc 2008.1 Incidence and mortality rates are rising in Asian countries. finger protein Y-linked (ZFY). The X-linked ZFX and Y-linked ZFY Older age, race and family history remain the only well-established C2H2-type genes are located at the very end of non-recombining risk factors and there are no established preventable risk factors regions of sex chromosomes in humans.8 The full-length ZFX for prostate cancer.2 Mutations of tumor suppressor genes in protein contains an acidic transcriptional activation domain, a individuals suffering from prostate cancer may lead its specificity nuclear localization sequence and a DNA-binding domain 9 among selected group of individuals. For example, it has been consisting of 13 C2H2-type zinc fingers. Despite many years of found that the putative tumor suppressor gene (PTEN/MMAC1) on relevant research, the physiological role of ZFX remains obscure. 10q23 is one of the most frequently deleted chromosomal regions However, Luoh et al.10 have found that in mouse embryonic and in human prostate cancer.3,4 Therefore, genome-based therapies adult hematopoietic stem cells, ZFX is required as a are potentially ideal options for prostate cancer therapy. transcriptional regulator for self-renewal in both stem cell types. Basic and applied research has gained new insights owing to Further research has found that deletion of ZFX abolished the the possibilities in site-specific manipulation of the mammalian self-renewal and decreased survival of hematopoietic stem cells, genome. This also can be effectively used in cancer therapy. Zinc causing a severe block in cell development.11 This evidence finger proteins are among the most abundant proteins in suggests that ZFX regulates the cell survival and activity, and it eukaryotic genomes with over 800 relevant proteins predicted could be used as a therapeutic target to treat human diseases. in human.5 They are involved in multiple functions, such as DNA Therefore, in this study, we investigated the effect of ZFX deletion recognition, RNA packaging, transcriptional activation, regulation on prostate cancer using cellular prostate cancer models: PC-3 of apoptosis, protein folding and assembly and lipid binding.6 and DU145. 1Department of Urology, Huashan Hospital, Fudan University, Shanghai, China; 2Institute of Urology, Fudan University, Shanghai, China and 3Department of Pathology, Huashan Hospital, Fudan University, Shanghai, China. Correspondence: Dr Q Ding, Department of Urology, Huashan hospital, Fudan University, No. 12, WuLuMuQi Zhong Road, Shanghai 200040, China. E-mail: [email protected] 4These two authors contributed equally to this work. Received 2 April 2012; revised 2 July 2012; accepted 4 July 2012; published online 17 August 2012 Knockdown of ZFX H Jiang et al 685 MATERIALS AND METHODS first-strand complementary DNA was synthesized from total RNA (5 mg) Immunohistochemistry using Super ScriptII reverse transcriptase (Invitrogen). ZFX mRNA expres- sion was evaluated by real-time PCR using SYBR Green Master Mix Kit on From 2006 to 2011, 45 prostate cancer samples were obtained from radical DNA Engine Opticon System (MJ Research, Waltham, MA). Glyceraldehyde- prostatectomy paired with adjacent normal tissues, which received detailed pathological assessment and regular follow-up. These samples 3-phosphate dehydrogenase (GAPDH) was applied as the input reference. The primers used for ZFX detection was 50-GGCAGTCCACAGCAAGAAC-30 were from a group of patients with prostate cancer in T1c-T2b, Gleason 0 0 ± À 1 and 5 -TTGGTATCCGAGAAAGTCAGAAG-3 . The primers used for GAPDH score 6–8 and mean prostate-specific antigen 15.3 8.9 ng ml .In 0 0 0 addition, 16 human benign prostatic hyperplasia (BPH) tissues were detection was 5 -TGACTTCAACAGCGACACCCA-3 and 5 -CACCCTGTTGCTG TAGCCAAA-30. For relative quantification, 2 À DDCT was calculated and used obtained by transurethral resection as normal control. The study was as an indication of the relative expression levels, which was calculated by conducted according to the regulations of the Ethics Committee of the 15 hospital. For immunohistochemical analysis, tissue specimens were fixed in subtracting CT values of the control gene from the CT values of ZFX. 10% formalin and embedded in paraffin. Tissue sections (4-mm) were deparaffinized and rehydrated. Endogenous peroxidase was blocked with Western blot 0.3% H2O2 in methanol. Following antigen retrieval, the sections were Total protein was isolated from cells that infected with lentivirus for 96 h. blocked with 5% bovine serum albumin for 20 min at room temperature Cells were washed with ice-cold phosphate-buffered saline and lysed in a and then probed with Rabbit Anti-ZFX Polyclonal Antibody (1:300 dilution; radioimmunoprecipitation assay buffer containing phenylmethyl sulfonyl- Sigma-Aldrich, Cat # HPA003877, St Louis, MO, USA) at 4 1C overnight. After fluoride and protease inhibitors on ice for 30 min. The lysates were clarified the incubation, the sections were treated with biotinylated goat anti-rabbit by centrifugation at 12 000 r.p.m. for 30 min at 4 1C. The total protein was immune-globulins at room temperature for 1 h, and visualized using the quantified by bovine serum albumin protein analysis method. Protein peroxidase-conjugated streptavidin and diaminobenzidine. Then the (20 mg) was loaded onto a 10% SDS-PAGE and transferred to polyvinyli- sections were counter stained with Mayer’s haematoxylin. The results dene difluoride membrane following electrophoresis (Millipore, Billerica, were evaluated with the help of two pathologists blinded to all clinical MA, USA). The proteins levels of ZFX, caspase-1, -3, -9 were detected by data. Immunopositivity was scored according to the percentage of positive respective antibodies using ECL kit (Amersham, Piscataway, NJ, USA) and cells in four distinct categories: 0 for 5%, 1 for 5–10%, 2 for 10–50% and exposed to X-ray film. GAPDH was used as loading control and detected by 3 for 50%. The staining intensity was then scored where 0 for negative, 1 an anti-GAPDH antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Bands for weak staining, 2 for intermediate staining and 3 for strong staining. on X-ray films were quantified with an ImageQuant densitometric scanner Both scores were added together, resulting in a maximum staining score of (Molecular Dynamics, Piscataway, NJ, USA). The antibodies used were 6 for any tissue score. 0–1, 2–3, 4–5 or 6 were considered negative ( À ), as follows: anti-ZFX (1:800 dilution; Sigma-Aldrich); anti-GAPDH (1:2000 weakly positive ( þ ), positive ( þþ) or hadro-positive ( þþþ) staining, dilution; Abcam, Cambridge, MA, USA); anti-caspase-1, anti-caspase-3, and respectively. anti-caspase-9 (1:1000 dilution; all from Abcam). Cell culture and cell proliferation analysis Formation of colonies Prostate cancer cells (PC-3 and DU145) and human embryonic kidney Lv-si-ZFX- or Lv-si-CTRL-infected cells (total of 800 cells per well) were (HEK) 293T cell line were obtained from American Type Culture Collection. seeded in six-well plates. The cells were grown by exchanging medium Cells were maintained in Dulbecco’s modified Eagle medium (Gibco, every 3 days until 2 weeks of culture. Then the cells were washed with Cambrex, MD) supplemented with 10% heat-inactivated fetal bovine phosphate-buffered saline and treated with Giemsa stain.
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