Lentivirus-mediated RNA Interference and Over-expression of CDK2AP1 CDNA Regulate CDK2AP1 Expression in Human Lung Cancer A549 Cells

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1 CHEM. RES. CHINESE UNIVERSITIES 2011, 27(3), Lentivirus-mediated RNA Interference and Over-expression of CDK2AP1 CDNA Regulate CDK2AP1 Expression in Human Lung Cancer A549 Cells GAO Nan 1, ZHANG Xing-yi 1, JIANG Rui 2, WANG Guan 3, LI Jin-dong 1, JIN Cheng-yan 1 and SUN Mei 4* 1. Department of Thoracic Surgery, the Second Hospital of Jilin University, Changchun , R. P. China; 2. Department of Orthopedic Surgery, China-Japan Union Hospital of Jilin University, Changchun , P. R. China; 3. Department of Cardiac Medicine, 4. Department of Pathology, the Second Hospital of Jilin University, Changchun , P. R. China Abstract Cyclin-dependent kinase 2-associated protein 1(CDK2AP1), a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, no studies on the function of CDK2AP1 in human lung cancer have been yet reported. In this study, overexpressing lentiviral vectors containing full-length CDK2AP1 cdna and CDK2AP1 shrna(short hairpin RNA) were constructed. Our results show that infecting A549 cells with lentivirus containing CDK2AP1 shrna or full-length CDK2AP1 cdna results in significantly down- or up-regulated the expression of CDK2AP1, respectively, thereby providing reliable tools for studying the function of CDK2AP1 in pulmonary carcinogenesis. Our data of MTT assay and flowcytometric analysis demonstrate that CDK2AP1 plays an important role in the proliferation/growth and cell cycling of A549 cells in vitro, and further investigation into its underlying mechanism of pulmonary carcinogenesis is needed. Keywords Lung; Cancer; CDK2AP1; shrna; Lentivirus Article ID (2011) Introduction Lung cancer is one of the human malignant tumors with the highest morbidity and mortality. There is a prevalence of more than 1.5 million cases worldwide per year [1], with high incidence especially in China. The aging population, the progression of urban industrialization as well as the contamination and destruction of human life environment result in the rising incidence. It has been estimated that the mortality and morbidity of lung cancer would continue to rise. The morbidity of lung cancer has ranked first in the incidences of various cancers in major cities of China. The developing knowledge of the molecular biology of tumor progression has revealed the changes of gene expression during lung cancer progression. And the elucidation of molecular mechanism under carcinogenesis would be of great importance in the prevention and treatment of cancers. Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), also known as DOC1, ST19, DORC1, DOC-1, or p12doc-1, was first isolated from normal keratinocytes as growth inhibitory factor by Todd et al. [2] in Located on human chromosome 12q24.31, the full length of CDK2AP1 gene is 1.6 kb, and its encoded protein is 12400(115 amino acids) [3,4]. Previous studies have demonstrated that the CDK2AP1 ectopic expression in keratinocytes is closely related to the increasing cell doubling time, implying its inhibitive effects on cell growth [5]. In this study, specific small interfering RNA(siRNA) of CDK2AP1 gene was designed and cloned into lentiviral vector, and lentiviral vector containing CDK2AP1 cdna was also constructed. The lentiviral particles were then packaged and used to infect lung cancer cell lines to test the silencing effects of RNA interference(rnai) and the over-expressing effects of lentiviral CDK2AP1 cdna, providing useful vectors for elucidating the function of CDK2AP1 in lung cancer development and progression. 2 Materials and Methods 2.1 Reagents Human embryonic kidney cells 293T and human lung cancer cell line A549 were purchased from Cell Bank of Chinese Academy of Sciences, Shanghai, China. The lentivirus plvthm, pspax2 and pmd2g vectors were obtained from Trono lab. Dulbecco s modified Eagle s medium(dmem) and *Corresponding author. sun.mei55@yahoo.com Received January 18, 2011; accepted March 21, Supported by the Grants from the Project of Scientific Innovation and Creative for Jilin Provincial Oversea Scholars(No ), Jilin Provincial Science and Technology Services(Nos , , , ), Jilin Provincial Development and Reformation Committee(No.2009Y042J12314), Jilin Province Talent Development Foundation (No.JRJB2007-2) and the Natural Science Foundation of China(Nos , ).

2 446 CHEM. RES. CHINESE UNIVERSITIES Vol.27 fetal bovine serum(fbs) were purchased from Gibco. The SYBR green PCR kit, plasmid extraction kit, Lipofectamine 2000,and 0.45 μm filter were obtained from TaKaRa, Qiagen, Invitrogen and Minipore, respectively. Short hairpin RNA(shRNA) and oligonucleotide primers were synthesized by Sangon Biotech(Shanghai) Co. Ltd. Mouse Anti-CDK2AP1 was obtained from Sigma-Aldrich. Goat Anti-mouse antibody was obtained from Santa Cruz Biotechnology, Inc. ECL+plus Western bloting Kit was purchased from Amersham. M-MLV and Trizol reagent were from Promega and Invitrogen, respectively. 2.2 CDK2AP1 sirna Design and plvthm- CDK2AP1 shrnas Construction The human CDK2AP1 cdna sequence(genebank accession number: NM_ ) was searched for suitable sirna target sequences, and three CDK2AP1 sirna sequences were designed by the Ambion sirna Target Finder. DNA oligos containing each of the target sequences were chemically synthesized, annealed, and inserted into the expression vector plvthm by double digestion with BamH I and EcoR I, and ligation with T4 DNA ligase in accordance with the manufacturer s guidelines. The ligation product was then transformed into competent Escherichia coli DH5αcells. As a control of CDK2AP1 sirna, a corresponding random sirna sequence was used. The targeting sequence of the short hairpin RNA(shRNA) was then confirmed by sequencing. 2.3 Construction of Overexpressing Vector plvthm-cdk2ap1 cdna CDK2AP1 cdna was obtained from plasmid containing CDK2AP1 cdna(from RZPD Library in German) by PCR, and then cloned into plvthm by double digestion with BamH I and EcoR I, and ligation with T4 DNA ligase in accordance with the manufacturer s guidelines. The ligation product was transformed into competent Escherichia coli DH5α cells. The recombinant was identified by PCR and confirmed by sequencing. 2.4 Selection of Effective CDK2AP1 shrnas Owing to the low transfection efficiency(<50%) of A549 cells, 293T cells were used to test the silencing efficiency of sirnas. The lentivirus vector plvthm-cdk2ap1 sirna and the overexpressing vector plvthm-cdk2ap1-cdna were co-transfected into 293T cells, respectively. The efficiency of knockdown was determined by Western blot. Briefly, 293T cells were rinsed with PBS and lysed with cooled 2 Lysis buffer(100 ml 1 mol/l Tris-HCl(pH=6.8), 2% 2-mercaptoethanol, 20% glycerol and 4% SDS on ice for min. After ultrasonic breakdown of cells, the cell lysate was centrifuged at 12000g and 4 C for 15 min. The supernatant with equal amounts of 2X loading buffer was then separated on 10% SDS polyacrylamide gel via a 5% polyacrylamide gel, and transferred onto PVDF membrane. The resulting Western blots were incubated with TBST containing 5% non-fat milk for 1 h at room temperature and then incubated with mouse Anti- CDK2AP1 antibody for 2 h at room temperature. The blots were then washed for 3 times with TBST and incubated with goat Anti-mouse antibody for 2 h at room temperature. After further washing, the blots were developed with the aid of ECL+plus Western blotting Kit. The blots were then applied to Kodak X-Omat film, and immunoreactive proteins were visualized on the developed film. 2.5 Lentiviral Particle Production and Titer Determination Lentiviral particles containing CDK2AP1 shrna and CDK2AP1 cdna were produced by cotransfection with viral particle packaging helper vector. 293T cells were transfected with Lipofectamine 2000 reagent according to the manufacturer s instructions. The viral supernatant was harvested at 72 h after transfection, passed through a 0.45 μm filter and supracentrifuged at r/min for 90 min. The supernatant was then discarded and the pellets were resuspended with cool PBS. Viral solution of 1 μl with a serial dilution was added into 293T cells, and the titer of viral particles containing CDK2AP1 shrna and CDK2AP1 cdna determined by limited serial dilution and real-time PCR at 48 h after transfection. 2.6 Detection of Knockdown Efficiency of CDK2AP1 shrna Human lung cancer cell line A549 cells were seeded in six-well plates to 80% 90% confluence. The viral supernatants containing CDK2AP1 shrna and CDK2AP1 cdna, respectively, were added into target A549 cells at the multiplicities of infection(5, 10, 20, 50 and 100). At 72 h after infection, total RNA was collected by means of Trizol reagent, and cdna was reversely transcribed with M-MLV. The expression level of CDK2AP1 mrna was analyzed by real-time quantitative reverse transcriptase PCR on a BioRad iq5 instrument. cdna was used for real-time quantitative RT-PCR via the SYBR green PCR master mixed with the β-actin as the control. The PCR conditions consisted of 40 cycles of pre-denaturation at 95 C for 15 s, then followed by denaturation at 95 C for 5 s, annealing and primer extension at 60 C for 30 s. RT-PCR results were expressed as fold changes in mrna expression with respect to those of the control cells. Target gene expression was normalized to the expression of the housekeeping gene β-actin for each sample. Data were analyzed via the 2-ΔΔCT method [6]. All reactions were performed in triplicate. CDK2AP1 protein expression was determined by Western blot as described previously. 2.7 MTT Assay Cell proliferation ability was assessed by methyl-thiazol tetrazolium(mtt) assay. Exponentially growing cells were inoculated into 96-well plates with cells per well, and after incubation for 24, 48, 72 and 96 h, 10 μl of sterile MTT [5 mg/ml, Sigma-Aldrich Corp] was added to each well. Following a further incubation at 37 C for 4 h, the reaction was

3 No.3 GAO Nan et al. 447 stopped by adding 100 μl of dimethyl sulfoxide(dmso). After thoroughly mixed for 10 min, the formazan production was determined by the measurement of the spectrometric absorbance at 490 nm on an enzyme immunoassay analyzer(1420 multi-label counter). The values obtained are proportional to the amount of viable cells. high as 90%(Fig.1), and Western blot results reveal that CDK2AP1 expression in cells transfected with plvthm-cdk2ap1 shrna-1 was the lowest compared to that of negative or mock control(fig.2), implying that CDK2AP1 shrna-1 was the most effective and the lentiviral vector itself didn t have specific inducing function. 2.8 Flow Cytometry Analysis of Cell Cycle The impact of CDK2AP1 silence and that of overexpression on A549 cell cycling were examined by FASC analysis. A549 cells were seeded into a 6-well plate, harvested at special time, and fixed in 70% alcohol. Percentage of A549 cells in each stage of cell cycle was determined by staining with propidium iodide(pi). The analysis of cell cycle distribution was performed on an FAC-Scan Flow Cytometer(Becton Dickinson, USA) in accordance with the manufacturer's guidelines, and each test was repeated three times. 2.9 Statistical Analysis Each experiment was performed at least three times, and data were expressed as mean±sd. Statistically significant differences between groups in each assay were determined by post-hoc Dunnett s t-test. The probability of <0.05 was considered to be statistically significant. 3 Results 3.1 Identification of Recombinant plvthm- CDK2AP1 cdna Fig.1 Flurescence images for Transfection of plvthm- CDK2AP1 shrna in 293T cells (A) Bright; (B) GFP. CDK2AP1 cdna was obtained by RT-PCR. The agar electrophoresis of PCR product and ligation product was conducted. The positive recombinant clones were then subjected to sequencing, and the blast of the sequencing data with those listed in NCBI revealed identical sequences, indicating that CDK2AP1 cdna was successfully inserted into the overexpressing vector. 3.2 Identification of the plvthm-cdk2ap1 shrnas The chemically synthesized shrnas were ligated to plvthm. The ligated plvthm-shrnas were then transformed into E. coli. PCR was used for the assay of positive clones. The clones containing shrnas were 320 bp in length, while clones non-containing shrnas were 282 bp. The sequences of positive clones were further confirmed by sequencing(data not shown), indicating the successful construction of plvthm-cdk2ap1 shrnas. 3.3 Identification of the Effective plvthm- CDK2AP1 shrnas The constructed plvthm-cdk2ap1 shrnas containing different targets as confirmed by sequencing, were transfected into 293T cells to identify the effective plvthm- CDK2AP1shRNA with the most knockdown efficiency. Fluorescence images shows that the transfection efficiency was as Fig.2 Efficacy of shrna-medicated suppression of CDK2AP1 CON: blank control; MOCK: negative control(transfected by negative shrna); 1# 3#: RNA interference groups(transfected by vectors containing different CDK2AP1 shrna). 3.4 Packaging Lentiviral Particles and Titer Assay plvthm CDK2AP1 shrna-1 and plvthm CDK2AP1 cdna were co-transfected into 293T cells with the helper packaging vectors, respectively. Green fluorescence was observed under a fluorescence microscope at 24 h after transfection. The supernatant was collected at 48 h after transfection and purified. The titer of lentiviral particles containing CDK2AP1 shrna-1 and plvthm CDK2AP1 cdna was TU/mL, and TU/mL as assayed by limited serial dilution and real-time PCR, respectively. 3.5 Optimal MOI Determination by Fluorescence Microscopy A549 cells were infected by lentiviral particles with

4 448 CHEM. RES. CHINESE UNIVERSITIES Vol.27 different MOIs(multiplicity of infection) after reaching 70% confluence. At 72 h after infection, fluorescence microscopy showed that the optimal MOI was 30(Fig.3). Fig.3 Fluorescence images of A549 cells infected by lentiviral particles (A) (C) Bright; (A ) (C ) GFP. 3.6 Effect of Lentiviral Particles Infection on CDK2AP1 mrna and Protein Expression As shown in Table 1, CDK2AP1 mrna level in cells infected by lentiviral particles containing CDK2AP1 cdna was 2.37 times more than that of the control group. While lentiviral particles containing CDK2AP1 shrna-1 had significant knockdown efficiency(61.4% when compared to the control group, P<0.05). Western blot assay revealed similar results as shown in Table 1. Table 1 Expression levels of CDK2AP1 mrna and protein detected by real-time PCR and Western blot in A549 cells of CON, CDK2AP1, RNAi( ) and RANi(+) groups, respectively Group a Relative CDK2AP1 mrna level(%) Relative CDK2AP1 protein level(%) CON 100.1± ±0.6 CDK2AP ±8.63 b 122.7±1.0 b RNAi( ) 100.2± ±0.1 RNAi(+) 38.6±6.3 c 25.6±0.7 c a. CON: untreated cells; RNAi( ): Scr-shRNA-Lv infected cells; RNAi(+): CDK2AP1-shRNA-Lv infected cells; CDK2AP1: CDK2AP1-Lv infected cells. b. P < 0.05 vs. CON or RNAi( ); c. P<0.05 vs. CON, RNAi( ) or CDK2AP Impact of Down- and up-regulated CDK2AP1 Expression on Growth and Cell Cycling of A549 Cells in vitro To explore the possible function of CDK2AP1 in the growth, proliferation and cell cycling of A549 cells, the growth dynamics and the cell cycling patterns of CON, CDK2AP, RNAi( ), and RNAi groups were determined by MTT assays and FACS Flow Cytometric analyses, respectively. MTT assays show that during a 96-h period, the growth of RNAi( )-A549 cells did not differ from its parent A549 cells(con-a549 cells). In comparison to those of CON-A549 cells and RNAi( )-A549 cells, the proliferation rate of A549 cells in RNAi(+)-A549 cells was significantly elevated, but it was significantly reduced in the CDK2AP1-A549 cells (Fig.4). Fig.4 A549 cell proliferation determined by MTT assay CON: untreated cells. RNAi( ): Scr-shRNA-Lv infected cells. RNAi(+): CDK2AP1-shRNA-Lv infected cells. CDK2AP1: CDK2AP1-Lv infected cells. * P<0.05 vs. CON or RNAi( ). # P<0.05 vs. CON, RNAi( ) or RNAi(+). FACS Flow Cytometric analysis shows that there is not difference in the G1/S ratio(an index of G1/S transition) between control-a549 cells(1.49±0.03) and si-control-a549 cells(1.57±0.05). Compared with those of CON-A549 cells and RNAi( )-A549 cells, the G1/S ratio in RNAi(+)-A549 cells(1.25±0.11) was significantly reduced whereas it was significantly elevated in the CDK2AP1-A549 cells(1.95±0.06). Therefore, these indicate that the modulation of CDK2AP1 expression by RNAi or overexpression lentiviruses results in the alteration of the proliferation/growth and cell cycling of A549 cells in vitro. 4 Discussion There is evidence to suggest that CDK2AP1 can inhibit the activity of CDK2-related kinases, leading to cell cycle arrest at DNA synthesis phase and apoptosis [6]. In addition, studies have shown that CDK2AP1 can interact with DNA polymerase α, regulate the phosphorylation of the large subunit p180 and negatively modulate DNA replication in S phase of cell cycle [7,8]. Lentiviral vector-mediated gene over-expression and gene silencing by RNAi have become a useful tool for studying molecular mechanism of cancers [9]. Our results show that infecting A549 cells with lentiviral particles containing CDK2AP1 shrna resulted in significantly down-regulated expression of

5 No.3 GAO Nan et al. 449 CDK2AP1, whereas A549 cells infected with lentiviral particles containing full-length CDK2AP1 cdna showed significantly up-regulated expression of CDK2AP1. There have been reports about CDK2AP1 gene function in human oral squamous cell carcinoma, colon and prostate cancer [10 12]. Recently, Zolochevska et al. [13,14] have reported that cell-cycle regulator CDK2AP1 suppresses malignant biological interactions between prostate cancer and bone cells, and modifies androgen-responsive pathway function. However, to date, the function of CDK2AP1 in human lung carcinogenesis remains unknown. Elucidation of the molecular mechanism of lung cancer is helpful for cancer gene therapy. In this study, we found that upregulated CDK2AP1 expression in A549 cells by CDK2AP1 overexpression lentivirus results in inhibited cell proliferation and growth accompanied by cell cycling arrest at G1-to-S stage, whereas down-regulated CDK2AP1 expression in A549 cells by CDK2AP1 RNAi lentivirus results in enhanced cell proliferation and growth accompanied by increased G1-to-S stage transition. Therefore, these indicate that modulation of CDK2AP1 expression by RNAi or overexpression lentiviruses results in the alteration of the proliferation/growth and cell cycling of A549 cells in vitro. In conclusion, this study represents on the construction of CDK2AP1 RNAi and over-expression lentiviruses for lung cancer. Our results show that infecting A549 cells with lentivirus containing CDK2AP1 shrna or full-length CDK2AP1 cdna resulted in significantly down-regulated or up-regulated CDK2AP1 expression, respectively. Therefore, these provide reliable tools for studying the function of CDK2AP1 in pulmonary carcinogenesis. In addition, our results demonstrate that CDK2AP1 plays an important role in the proliferation/growth and cell cycling of A549 cells in vitro, and further investigation into its underlying mecha- nism of pulmonary carcinogenesis is needed. References [1] Stewart B.W., Kleihues P., World Cancer Report, IARC Press, Lyon, 2003, 182 [2] Todd R., McBride J.,Tsuji T., Donoff R. B., Nagai M., Chou M. Y., Chiang T., Wong D. T., FASEB J., 1995, 9, 1362 [3] Tsuji T., Duh F. M., Latif F., Popescu N. C., Zimonjic D. B., McBride J., Matsuo K., Ohyama H., Todd R., Nagata E., Terakado N., Sasaki A., Matsumura T., Lerman M. I., Wong D. T., J. Biol. Chem., 1998, 273, 6704 [4] Figueiredo M. L., Kim Y., St John M. A., Wong D. T., Clin. Cancer Res., 2005, 11, 3939 [5] Sherr C. J., Science, 1996, 274, 1672 [6] Shintani S., Ohyama H., Zhang X., Mcbride J., Matsuo K., Tsuji T., Miao F. G., Hu G. F., Kohno Y., Lerman M., Todd R., Wong D. T. W., Mol. Cell. Biol., 2000, 20, 6300 [7] Matsuo K., Shintani S., Tsuji T., Nagata E., Lerman M., Mcbride J., Nakahara Y., Ohyama H., Todd R., Wong D. T. W., FASEB J., 2000, 14, 1318 [8] Hu M. G., Hu G. F., Kim Y., Tsuji T., McBride J., Hinds P., Cancer Res., 2004, 64, 490 [9] Peng H., Shintaniy S., Kim Y., Wong D. T. W., Neoplasia, 2006, 8, 1028 [10] Yuan Z., Kent T. S., Weber T. K., Oncogene, 2003, 22, 6304 [11] Shin J. M. D., Yuan Z. Q., Fordyce K., Sreeramoju P., Kent T. S., Kim J., Wang V., Schneyer D., Weber T. K., Surgery, 2007, 142, 222 [12] Elizabeth H. G., Sofiya N. M. V., Curr. Drug. Discov. Technol., 2010, 7(2), 86 [13] Zolochevska O., Figueiredo M. L., Prostate, 2010, 71, 353 [14] Zolochevska O., Figueiredo M. L., Prostate, 2009, 69, 1586