In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-014-9838-y

REPORT

Overexpression of Lhx8 inhibits cell proliferation and induces cell cycle arrest in PC12 cell line Haoming Li & Jianbing Qin & Guohua Jin & Linqing Zou & Jinhong Shi & Xiao Han & Xiang Cheng & Xinhua Zhang

Received: 15 July 2014 / Accepted: 20 October 2014 / Editor: T. Okamoto # The Society for In Vitro Biology 2014

Abstract LIM-homeobox genes play a pivotal function in tissue patterning and differentiation, Lhx8 is a member of LIM-homeobox gene family, and it is selectively expressed in embryonic basal forebrain and is a key factor for the determination of cholinergic cells fate. However, besides cholinergic differentiation, little is known about the potential role of Lhx8 in cell biology. In this study, we transfected Lhx8 complementary DNA (cDNA) into PC12 cell line using lentiviral vectors to acquire the cells which stably expressed high level of Lhx8, and we provide the experimental evidence that overexpression of Lhx8 inhibits cell proliferation and induces cell cycle arrest but not apoptosis in vitro. In conclusion, besides cholinergic differentiation, our results suggest that Lhx8 also plays as a suppressor gene of proliferation in cell biology. Keywords Lhx8 . PC12 . Proliferation . Cell cycle

Introduction LIM-homeobox genes encode LIM-homeodomain proteins; they play a pivotal function in tissue patterning and differentiation, especially in neural tissues development of the central nerves system (CNS) (Zhao et al. 1999; Srivastava et al. 201010, Simmons et al. 2012). Lhx8, also called L3 or H. Li : J. Qin : G. Jin (*) : L. Zou : J. Shi : X. Han : X. Cheng : X. Zhang Department of Human Anatomy, the Jiangsu Key Laboratory of Neuroregeneration, Medical School of Nantong University, 19 Qixiu Road, Nantong 226001, Jiangsu, China e-mail: [email protected] G. Jin : L. Zou : J. Shi : X. Han Department of Human Anatomy and Histoembryology, Medical School of Soochow University, Suzhou 215123, China

Lhx7, is a member of LIM-homeobox gene family; Lhx8 encodes a highly conserved LIM-homeodomain (LIM-HD) protein between mouse and humans; and it is selectively expressed in embryonic medial ganglionic eminence (MGE) and is a key factor for the determination of cholinergic cells fate (Zhao et al. 2003; Mori et al. 2004; Manabe et al. 2005). Lhx8 promotes the expression of choline acetyltransferase (ChAT) and vesicular Ach transporter (VAChT), the basal forebrain cholinergic neurons are decreased in Lhx8-null mice, and Lhx8-knockdown embryonic stem cells specifically lost the cholinergic phenotype (Manabe et al. 2007, 2008; Li et al. 2014). Lhx8 is also preferentially expressed in oocytes and germ cells; it has been implicated in folliculogenesis and is a critical factor for maintenance and differentiation of the oocyte during early oogenesis; Lhx8-null mice are infertile due to lack of oocytes; and transition of primordial follicles into primary follicles is impaired (Pangas et al. 2006; Choi et al. 2008). Lhx6 is another member of LIM-homeobox genes; it is required for radial migration of interneurons and specification of GABAergic neuronal differentiation (Grigoriou et al. 1998; Flandin et al. 2011); and recently, study shows that Lhx8 and Lhx6 coordinately induce neuronal expression of Sonic hedgehog (Shh) that controls the generation of interneuron progenitors in MGE (Flandin et al. 2011). Proteins containing LIM-domain are emerging as key factors in human cancers. All members of human LIM-domainonly (LMO) proteins are implicated in the progression of several cancers (Zhou et al. 2012; Matthews et al. 2013); LIM-homeobox transcription factor alpha (LMX1A) inhibits tumourigenesis, epithelial-mesenchymal transition, and stemlike properties of epithelial ovarian cancer (Chao et al. 2013); knockdown of Lhx6 increases cell proliferation, inhibits apoptosis, and induces cell cycle arrest in lung cancer tissues and cell lines; and Lhx6 may be a putative tumor suppressor gene in lung cancer (Liu et al. 2013).

LI ET AL.

To better understand the function of Lhx8, in this report, we transfected Lhx8 complementary DNA (cDNA) into pheochromocytoma (PC12) cell line using lentiviral vectors to acquire the cells which stably expressed high level of Lhx8. Using this cell model, we provided the experimental evidence that overexpression of Lhx8 inhibited cell proliferation and induced cell cycle arrest but not apoptosis in vitro.

Materials and Methods Establishment of stable PC12 cell line. Lhx8 overexpression lentivirus (LV-Lhx8) and negative control lentivirus (LV-NC) were acquired as our previous description using a GV287 lentiviral expression system (Genechem, Shanghai, China) (Zhu et al. 2013); the titer of LV-NC and LV-Lhx8 was 1× 109 and 2×108 TU/ml, respectively. Differentiated PC12 cell line was obtained from Cell library of Chinese Academy of Science (Shanghai, China) and plated at a density of 1×105 cells on 6-well plates with DMEM/F12 containing 10% fetal bovine serum (FBS) as expansion medium (2 ml/well). After incubation for 24 h, the culture medium was replaced by expansion medium containing 20 μl 2×108 TU/ml LV-Lhx8 or 4 μl 1×109 TU/ml LV-NC with 5 μg/ml ploybrene (1 ml/ well). Following incubation for 48 h, the culture medium was replaced by fresh expansion medium without lentivirus. Seventy-two hours later, for selection of stable transformants, the transfected cells were maintained in expansion medium containing 100 μg/ml G418 (Sangon, Shanghai, China) (2 ml/ well) for 72 h; then the expansion medium containing 50 μg/ml G418 (2 ml/well) was maintained for another 2 wk; and the medium was exchanged every 48 h. After 2 wk of culture, the stable cell lines were incubated in accutase (Sigma, St. Louis, MO) for 5 min and gently triturated into single-cell suspensions; then the cells suspension was maintained at a density of 1×105 cells/ml into 25-ml culture flasks; and the cultured cells were harvested at 80% confluence for other experiments. Microscope and Immunohistochemistry assay. LV-NC and LV-Lhx8 PC12 stable cells (LV-NC group and LV-Lhx8 group) as well as wild-type (WT) PC12 cells (WT group) were seeded on coverslips in 24-well plates with expansion medium (200 μl/well) at 80% confluence; then all cells were fixed in 4% paraformaldehyde for 10 min; and cells nuclei were counterstained with Hoechst (Sigma). EGFP fluorescence of cells was investigated using Olympus laser confocal microscope (Fv10i, Olympus, Tokyo, Japan). In immunohistochemistry assay, cells in different groups were seeded at a density of 5×104 cells in 24-well plates for 24 h; then the cells were fixed in 4% paraformaldehyde for 10 min and blocked using 5% BSA for 1 h; rabbit antiCaspase-3 (active form, 1:100) (Millipore, Massachusetts,

USA) was incubated overnight at 4°C; then the cells were incubated with secondary antibodies conjugated to fluorescein 594 for 2 h at room temperature; and cell nuclei were counterstained with Hoechst (Sigma). Caspase-3-positive cells were investigated using Olympus laser confocal microscope (Olympus). Western blot. Cells in different groups were cultured in 6-well plates with expansion medium (2 ml/well) at 80% confluence, and total protein was extracted using RIPA Lysis Buffer (Beyotime, Jiangsu, China), and the protein concentration was determined using Bradford Protein Assay Kit (Beyotime). Equal amounts of protein resolved on SDSpolyasrylamide gel electrophoresis (PAGE) using 10% separation gel. Gels were transferred to a PVDF membrane using BioRad Semi-Dry Transfer Cell (BioRad, Hercules, CA, USA) at 15 V for 20 min, and then blocked with 5% milk in TBST buffer and incubated with primary antibody rabbit antiLhx8 (1:200; Abcam, Cambridge, UK) and mouse anti-βactin (1:2000; Beyotime) overnight at 4°C. The membrane was subsequently incubated with HRP-linked goat anti rabbit or mouse IgG. The final detection of Lhx8 and β-actin was carried out with the use of ECL-plus regent (BioRad). Cell proliferation assay. Cells in different groups were seeded in a 96-well plate at a density of 1×104 cells/well. Cell viability was determined after 24, 48, and 72 h, respectively, using Cell Counting Kit-8 (CCK-8) (Beyotime, Jiangsu, China) according to the manufacturer’s instructions, and the OD value at 450 nm was detected using Synergy 2 enzyme mark instrument (BioTek, Winooski, Vermont). EdU and BrdU assay. Cells at S phase were measured by 5ethynyl-2′-deoxyuridine (EdU) incorporation assay using EdU assay Kit (Ribobio, Guangzhou, China). Briefly, cells in different groups were seeded in a 24-well plate at a density of 5×104 cells/well. Twenty-four hours later, all cells were exposed to EdU (50 μm) for 2 h, followed by staining with Apollo-567 reaction regent cocktail for 30 min; the cells nuclei were counterstained with Hoechst (Sigma). The EdUlabeled cells were examined under Olympus laser confocal microscope (Olympus). For BrdU incorporation assay, cells in different groups were seeded in a 24-well plate at a density of 5×104 cells/ well for 24 h; then all cells were exposed to BrdU (10 μm) (Sigma) for 2 h. After fixed in 4% paraformaldehyde for 10 min, all cells were incubated in 2 M HCl for 15 min and 0.1 M borate buffer for 10 min to neutralization. Then, all cells were blocked for 1 h in 5% FBS, primary antibodies were added and incubated overnight at 4°C; and antibodies were mouse anti-BrdU (1:100, Sigma) and rabbit anti-Lhx8 (1:300, Abcam). Then, the cells were incubated with secondary antibodies conjugated to fluorescein 594 and 647 for 2 h at room

LHX8 INHIBITS CELL PROLIFERATION IN PC12

temperature. The BrdU-labeled cells were investigated using Olympus laser confocal microscope (Olympus). FACS assay In cell cycle assay, 5×105 cells were seeded into 25-ml culture flasks for 24 h. Then, the cells in different groups were harvested and fixed in 70% ice-cold ethanol for 2 h; cells were stained by propidium iodide (PI) using BD Cycletest™ Plus DNA Reagent Kit (BD Biosciences, Franklin Lakes, NJ). In apoptosis assay, cells were harvested and stained by Annexin V using Annexin V-PE Apoptosis Detection Kit (Beyotime). Cells at different phases and the apoptotic cells were assessed using a FACS Calibur Instrument (BD Biosciences). Real-time PCR. Cells in different groups were cultured in 6well plates with expansion medium (2 ml/well) at 80% confluence, and the total RNA was isolated using UNIQ-10 Spin Column RNA Purified Kit (Sangon, Shanghai, China). The first strand cDNA was synthesized using RevertAidTM First Strand cDNA Synthesized Kit (Fermentas, Burlington, Canada). First Strand cDNA was subsequently subjected to Corbett RG-6000 PCR system (QIAGEN, Dusseldorf, German) using FastStart Universal SYBR Green Master Mix (Roche, Basel, Switzerland). The reactions were optimized by varying the annealing temperatures at 52~55°C; the sense and antisense primers were synthesized as follows: GAPDH 5′GCAAGTTCAACGGCACAG-3′, 5′-GCCAGTAGACTC CACGACAT-3′; c-myc 5′- AGTCAGGGTCATCCCCAT CA-3′, 5′-TGGAGCATTTGCGGTTGTTG-3′; p21 5′CTCAGAGCCACAGGCACCAT-3′, 5′-TCAAAGTTCCAC CGTTCTCG-3′; p53 5′-AGCTCCAGTTCATTGGGACTTA T-3′, 5′-TGACACCCTGCTGGGAAGTAG-3′; and cyclinD1 5′-CCTGACTGCCGAGAAGTTGT-3′, 5′-TCATCCGCCT CTGGCATTTT-3′. Statistical analysis Data from the experiments were subjected to Student’s t test or one-way ANOVA test using SPSS 11.5 statistical software; all data were represented as the mean± SEM; and all experimental data were obtained from a minimum of three independent experiments.

Results PC12 cells stably expressing Lhx8-EGFP (LV-Lhx8 group) or EGFP alone (LV-NC group) were obtained after lentivirus transfection. In WT groups, no green fluorescence was founded (Fig. 1A~D); after 2-wk selection by G418, ~95% cells in LV-NC group (Fig. 1E~H) and LV-Lhx8 group (Fig. 1I~L) expressed EGFP fluorescence, respectively; the difference between two groups was not statistically significant (Fig. 1M). However, Lhx8 protein level in LV-Lhx8 group

was obviously higher than that in WT group and LV-NC group (Fig. 1N). To evaluate the function significance of Lhx8 in PC12 cells, we examined growth suppression by Lhx8 overexpression using CCK-8 assay; the OD value (450 nm) in LV-Lhx8 group was significantly lower than that in WT group or LVNC group at 24, 48, and 72 h, respectively (Fig. 1O); the results of the cell proliferation assay indicated that ectopic expression of Lhx8 inhibited the growth of PC12 cells compared with that in the control groups. We next determined whether inhibition of cell growth by Lhx8 was related to the cell cycle arrest and/or apoptosis. First, the effect of Lhx8 expression on the cell cycle was evaluated by FACS assay (Fig. 2A~D). In G1 phase, the percentage of cells in LV-Lhx8 group was obviously higher than that in WT group or LV-NC group (Fig. 2D); in S and G2 phase, the percentage of cells in LV-Lhx8 group was obviously less than that in WT group or LV-NC group (Fig. 2D); the cells percentage in S phase was also measured by EdU incorporation assay (Fig. 2E~H) and BrdU immunohistochemistry (Fig. 2I~L); the percentage of cells at S phase in LV-Lhx8 group was also less than other groups (Fig. 2H, L). The cells in LV-Lhx8 group displayed a significantly higher frequency at G1 phase and a lower frequency at S or G2 phase. These results demonstrated a reduction in the percentages of cells at S or G2 phase and retention at G1 phase after ectopic Lhx8 expression. In apoptosis assay, the proportion of Annexin V positive cells or Caspse-3-labeled cells were detected using FACS (Fig. 3A~D) and immunohistochemistry assay (Fig. 3E~H), respectively. However, the results revealed that there was no significant difference in three groups (Fig. 3D~H). Briefly, Lhx8 expression did not trigger apoptosis of PC12 cells in our experimental system. Therefore, the results suggested that inhibition of cell growth by Lhx8 was related to the cell cycle arrest but not apoptosis in vitro. To identify the Lhx8-downstream genes that may concern the regulation of PC12 cell proliferation and cell cycle arrest, c-myc, p21, p53, and cyclinD1 were investigated using realtime PCR assay. We found the c-myc gene in LV-Lhx8 group was obviously decreased than those in WT group and LV-NC group (Fig. 4A). However, p21 and p53 in LV-Lhx8 group were slightly higher than those in other groups (Fig. 4B, C), and cyclinD1 was slightly decreased than others (Fig. 4D); the difference of p21, p53 and cyclinD1 in three groups were not statistically significant.

Discussion LIM-homeobox genes play a key function in neural tissue development of CNS, these genes encode LIM-homeodomain proteins, and LIM-homeodomain proteins are regulatory

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Figure 1. No green fluorescence was founded in WT group (A~D), ~95% cells in LV-NC group (E~H) and LV-Lhx8 group (I~L) expressed EGFP fluorescence, the difference between two groups was not statistically significant (M), and scale bar=50 μm. The Lhx8 protein expression

in LV-Lhx8 group was higher than others (N) (***P