ORIGINAL RESEARCH ARTICLE

Journal of

Cellular Physiology

Oral Squamous Cancer Cell Exploits hnRNP A1 to Regulate Cell Cycle and Proliferation CHENG YU,1 JIHUA GUO,1 YU LIU,2 JUN JIA,1* RONG JIA,1*

AND

MINGWEN FAN1*

1

Hubei-MOST KLOS & KLOBME, School & Hospital of Stomatology, Wuhan University, Wuhan, PR China

2

College of Life Sciences, Wuhan University, Wuhan, PR China

Oral squamous cell carcinoma (OSCC) is a common human malignant tumor with high mortality. So far, the molecular pathogenesis of OSCC remains largely unclear. Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an important multi-function splicing factor and closely related to tumorigenesis. hnRNP A1 is overexpressed in various tumors, and promotes aerobic glycolysis and elongation of telomere, but the function of hnRNP A1 in cell cycle and proliferation remains unclear. We found that hnRNP A1 was overexpressed in OSCC tissues, and was required for the growth of OSCC cells. Moreover, hnRNP A1 was highly expressed in the G2/M cell cycle phase. Knockdown of hnRNP A1 induced G2/M arrest. DNA microarray assay result showed that hnRNP A1 regulated the expression of a number of target genes associated with G2/M phase. Moreover, hnRNP A1 controlled the alternative splicing of CDK2 exon 5. These findings suggested that hnRNP A1 plays key roles in the regulation of cell cycle progression and pathogenesis of OSCC. J. Cell. Physiol. 230: 2252–2261, 2015. © 2015 Wiley Periodicals, Inc.

Previous studies have described heterogeneous nuclear ribonucleproteins (hnRNPs) as a group of chromatinassociated RNA-binding proteins (Dreyfuss et al., 1993). More than 20 kinds of hnRNPs are present in human cells, (A–U), most of which bind to RNA directly. The most abundant and ubiquitously expressed proteins among these hnRNPs are the six core proteins, namely, A1, A2, B1, C1, B2, and C2 (Dreyfuss et al., 1993). These proteins can indiscriminately bind to nascent RNA polymerase II transcripts, and then convert heterogeneous nuclear RNAs into hnRNP particles (Krecic and Swanson, 1999). hnRNP A1 is probably the most-studied and best-known hnRNP. It contains 320 amino acid residues, and has a molecular weight of 34 kD (Jean-Philippe et al., 2013). hnRNP A1 has two N-terminal RNA recognition motifs and one Cterminal glycine rich domain (Cartegni et al., 1996). It participates in several RNA-related processes, such as RNA binding, alternative splicing, and mRNA transport. Specifically, hnRNP A1 can regulate the binding of small nuclear ribonucleoproteins and other RNA processing factors with mRNA precursors, which affects the splicing process of mRNA precursors (Burd and Dreyfuss, 1994). For example, hnRNP A1 can affect a series of pre-mRNA 50 splice site selection and mutually exclusive exon selection (Mayeda and Krainer, 1992; Mayeda et al., 1993; Clower et al., 2010). Moreover, hnRNP A1 shuttles between the nucleus and cytoplasm and associates with mRNA throughout the cytoplasm (Twyffels et al., 2011). Therefore, hnRNP A1 may somehow be involved in mRNA export. hnRNP A1 is closely related to tumorigenesis. Studies have shown that hnRNP A1 is overexpressed in various tumor tissues, including lung cancer (Pino et al., 2003), Burkitt lymphoma (Brockstedt et al., 1998), multiple myeloma (Shi et al., 2008), and leukemia (Iervolino et al., 2002), but is expressed at low levels in normal tissues. Moreover, hnRNP A1 can elongate telomere by activating telomerase, which induces the occurrence and development of malignant tumor (Ting et al., 2009). hnRNP A1 enhances the switching from PKM1 to PKM2 of pyruvate kinase type K, which promotes aerobic glycolysis and facilitates tumor formation (Chen et al., © 2 0 1 5 W I L E Y P E R I O D I C A L S , I N C .

2010; Clower et al., 2010). The alternative splicing event controlled by hnRNP A1 sheds light on the molecular mechanism by which hnRNP A1 facilitates tumorigenesis. However, whether and how hnRNP A1 regulates cell proliferation in cancer remain unclear. Although considerable research has revealed the association of hnRNP A1 and cancer, several studies have shown that hnRNP A1 is not involved in the growth of certain cancer cells (Ben-David et al., 1992; Patry et al., 2003; He et al., 2005). The relationship between hnRNP A1 and oral cancer remains unclear. In the present study, we examined the clinicopathologic and biological significance of hnRNP A1 in oral squamous cell carcinoma (OSCC). Our results showed that hnRNP A1 was overexpressed in OSCC tissues, and was required for the growth of OSCC cells. Moreover, hnRNP A1 was highly expressed in the G2/M cell cycle phase and regulated a number of target genes associated with the G2/M phase. These findings suggest hnRNP A1 plays key roles in the pathogenesis of OSCC through the regulation of cell cycle progression.

Cheng Yu and Jihua Guo contributed equally to these work. Conflict of interest: None. Contract grant sponsor: National Science Foundation of China; Contract grant number: 81271143. Contract grant sponsor: Fundamental Research Funds for the Central Universities; Contract grant number: 2042014kf0270. *Correspondence to: Mingwen Fan, Rong Jia, and Jun Jia, 237 Luoyu Road, Wuhan City 430079, PR China. E-mails: [email protected]; [email protected]; [email protected] Manuscript Received: 25 June 2014 Manuscript Accepted: 5 February 2015 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 9 March 2015. DOI: 10.1002/jcp.24956

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Methods and Materials Cells

Sampling of normal gingival tissues from donors who received forced eruption operation or OSCC tissues was approved by the Ethics Committee at the School of Stomatology in Wuhan University. Normal gingival epithelial cells (N1, N2, N3, and N4) were dissociated from gingival tissues and grown in keratinocyte growth medium (Lonza, Basel, Switzerland). Cells were collected upon reaching 70% confluence. Primary OSCC cells (T1, T2, and T3) were cultured in Dulbecco’s modified Eagle medium (DMEM, HyClone, Beijing, China) supplemented with 10% fetal bovine serum (FBS) and 1% antibioticantimycotic (Gibco-BRL, Grand Island, NY). Cancer cells were purified by removing fibroblasts with short treatment of 0.25% trypsin-EDTA (Invitrogen, Carlsbad, CA). CAL 27 (an OSCC cell line) and HEK-293 cells were grown in DMEM with 10% FBS and 1% antibiotic-antimycotic. Plasmids

The human CDK2 or hnRNP A1 gene was reverse-transcribed and amplified from CAL 27 cells using primers 50 GAATTCCGCTTCATGGAGAACTTCCA-30 and 50 GGATCCCAGTCGAAGATGGGGTACTGGC-30 , or 50 CGAAGAAGCATCGTTAAAGTCTCTC-30 and 50 CTTCTCTGGCTCTCCTCTCCTGC-30 . A CDK2 isoform 1 or 2 gene fragment was fused to green fluorescent protein (GFP) gene in pEGFP-N1 (Clontech, Palo Alto, CA), a eukaryotic expression plasmid. A T7-Tag (Jia et al., 2010) was added to the 50 of hnRNP A1 gene by overlapping PCR. The T7-A1 fragment was then cloned into pLVX-IRES-Puro vector (Clontech) at EcoRI and NotI sites to generate a T7-tagged hnRNP A1 overexpression plasmid (named T7-A1). Tissue microarray and immunohistochemistry

A human OSCC tissue microarray, including 49 OSCC and 10 normal oral mucosal tissues, was obtained from Alenabio Co., Ltd. (Xi’an, China). Among the 49 OSCC tissues, nine cases are grade I, 32 are grade I–II or II, 4 are grade II–III or III, and 4 have no grade information. The avidin–biotin peroxidase complex method was used for immunohistochemical staining in the human OSCC tissue microarray with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA). The monoclonal mouse anti-hnRNP A1 (clone 4B10) was obtained from Santa Cruz Biotechnology, Dallas, TX. A1 specific staining intensity was divided into four levels (0–3). Cell numbers in each level were counted and the percent of total (0–100) was calculated. Staining scores were calculated by multiplying the intensity score (0–3) by the total percentage. RNAi

Human hnRNP A1 siRNA #1 and #2 were obtained from Sigma-Aldrich Co. LLC. (SASI_Hs02_00333298; SASI_Hs02_00333299; Sigma-Aldrich, St. Louis, MO). Nonspecific (NS) siRNA was obtained from Santa Cruz Biotechnology. Inc. (sc-37007). 5  105 CAL 27 or T3 cells were seeded into a six well plate and transfected with 10 nM hnRNP A1 or NS siRNA in the presence of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 48 h, cells were passed and received another transfection. After 96 h, the number of cells were counted. Total protein and RNA were also collected. Western blot

Total protein samples were separated in 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane JOURNAL OF CELLULAR PHYSIOLOGY

was blotted with the following antibodies: rabbit polyclonal antiCDK2 (Upstate, Charlottesville, VA), mouse monoclonal antihnRNP A1 (Santa Cruz Biotechnology), mouse monoclonal anti-GFP (Abmart, Shanghai, China), or horseradish peroxidaselabeled mouse anti-b-actin antibody (Sigma-Aldrich). Cell cycle analysis

CAL 27 or T3 cells were treated with hnRNP A1 siRNA #1 or NS siRNA twice in a 48-hour interval. After 96 h, cells were collected and prepared with COULTER DNA PREP Reagent Kit (Beckman Coulter, Miami, FL) according to the manufacturer’s instruction. Cell cycles were analyzed using a BD Biosciences FACSCalibur flow cytometer and Modfit LT software (Verity Software House, Topsham, ME). Cell-cycle fractionation was performed by flow cytometry with Hoechst 33342 staining. In brief, cells were incubated with 20 mM Hoechst 33342 for 1 h, and then sorted by a Beckman Coulter MoFloTM XDP cell sorter. Cells in different cell cycle stages were separated based on their fluorescent density. Cells in G1/G0 or G2/M stage were identified based on 2N and 4N DNA peaks. Cells between 2N and 4N peaks were regarded as S stage. Total RNA and proteins were immediately collected after sorting. DNA microarray assay

CAL 27 cells were treated with 10 nM hnRNP A1 siRNA #1 or NS siRNA twice in a 48-hour interval. Each treatment was performed triply. After 96 h, total RNA was collected and reverse transcribed. The cDNA was labeled with NimbleGen One-Color DNA Labeling Kit and hybridized with NimbleGen 12  135 K Human Gene Expression Microarrays, which measured the expression levels of 45,033 genes. After washing, arrays were scanned with Axon GenePix 4000B scanner. Data were extracted and normalized using NimbleScan v2.5 Software. Differentially expressed genes with statistical significance were identified through Volcano Plot filtering. Data were submitted to GEO database in NCBI, with access number of GSE57819. RNA preparation and reverse transcriptase PCR (RTPCR)

Total RNA from cells was extracted using Total RNA Miniprep Kit (Axygen Scientific, Hangzhou, China). One microgram of total RNA was treated with DNase I (Invitrogen), and then reverse transcribed using random hexamers at 37°C with the Superscript II reverse transcriptase kit (Invitrogen) and amplified using the following primer pairs: 50 -CGAAGAAGCATCGTTAAAGTCTCTC-30 and 50 CTTCTCTGGCTCTCCTCTCCTGC-30 for hnRNP A1, 50 TGGACTAGCCAGAGCTTTTGGAG-30 and 50 -ACTTGGCTTGTAATCAGGCATAGAAG-30 for CDK2, 50 -ACAATCCAGCTAATCCAGAACCACT-30 and 50 GGTCTTGGCTGAGGTTTCAATG-30 for NRAS, 50 CACGCCAACATCGTTACGCT-30 and 50 -TGGGATTGACTTGGCTCGG-30 for PCTK1, 50 -CCTGGAGGAACGGAGACAAAGGAG-30 and 50 -GAAGAGCGTGTTGACCAGCGTTGA-30 for SEPT3, 50 -CTCGCTCTTCGACTACCATGACACAAG-30 and 50 -CATCATCCGTGGGGAACTGGTAGATC-30 for SEPT8, 50 -CTGACCTTGAGCATGACCAGACCATG-30 and 50 -GAAGCCTCCAAGCATTCTGTCCCATAC-30 for CLASP1, 50 -CTCTGAAACATGGTGGCCTGAGAGA-30 and 50 -CCGAGGGTAACCAAATGGTCCAG-30 for PARD3B, 50 -CCAAGAGGCAGCTAAGTGTGTGAGTG-30 and 50 -GAACTTGACAATGGCTGGGTGGTC-30 for NEK11, 50 -GAGAAGGAACGTCGTGATGCCTTG-30 and 50 -GTTCCCACAAGCGTCTCAGCCAT-30 for NEK9, 50 -ATCACCGAACCC-

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Fig. 1. hnRNP A1 is overexpressed in OSCCs. (A–C) The expression of hnRNP A1 was analyzed in a tissue array (including 49 OSCC tumor samples and 10 normal oral mucosal samples) using immunohistochemistry. A: Representative immunohistochemical staining results of hnRNP A1 in normal oral mucosal epithelium, or OSCCs with different grades. B: Box plot of immunostaining scores of hnRNP A1 in normal or tumor tissues of the tissue array. C: Box plot of immunostaining scores of hnRNP A1 in tumor tissues with grade I (9 cases), grade I–II and II (32 cases), or grade II–III and III (4 cases). Four cases in the array have no grade information. D: Western blot analysis of the expression of hnRNP A1 in primary human oral squamous cancer cells, CAL 27 cells, and normal primary oral mocosal epithelial cells. b-actin served as the loading control.

GAGCACACCA-30 and 50 -CTAGGGGCTTCTACTCTCTTGGTGTCAC-30 for RCC2, 50 -TTGAGGAGTGCTGTTTCCGCAG-30 and 50 -GTGGACTGCTTCCAGGTGTCATATTG-30 for IGF2, 50 -GCTGGTGAAGATGGAGTTTGACGAG-30 and 50 -GTACTCCTTGGAGCCGCCTTTCAT-30 for DNM2, 50 -GTCATCAATGGAAATCCCATCACC-30 and 50 TGAGTCCTTCCACGATACCAAA-30 for GAPDH. Real-time quantitative RT-PCR (qRT-PCR)

Real-time qRT-PCR was performed three times using SYBR Green (Roche, Mannheim, Germany) and a 7900HT Fast RealTime PCR machine (Applied Biosystems, Foster City, CA). Primers of CLASP, NEK9, NEK11, NRAS, RCC2, DNM2, JOURNAL OF CELLULAR PHYSIOLOGY

PCTK1, and SEPT8 were purchased from GeneCopoeia (Guangzhou, China). All other specific primers for qRT-PCR are listed as follows: 50 -GAAGGTGAAGGTCGGAGTC-30 and 50 -GAAGATGGTGATGGGATTTC-30 for GAPDH; 50 -CCCAGCACAATGAAGATCAA-30 and 50 -ACATCTGCTGGAAGGTGGAC-30 for b-actin; 50 -AAGCAATTTTGGAGGTGGTG30 , and 50 -ATAGCCACCTTGGTTTCGTG-30 for hnRNP A1; 50 -CGCTGGTCAACACGCTCTT-30 and 50 -CATGCCCGATAGCTTTGATCT-30 for SEPT3; 50 -GTTCGTACTTACACCCATGAGGTGACTC-30 (exon 4 and 6 junction primer) and 50 -GAAACTTGGCTTGTAATCAGGCATAGAAG-30 (exon 6) for exon 5-skipped CDK2; 50 -TTGGACTAGCCAGAGCTTTTGGAGTC-30 (exon 4) and 50 -

hnRNP A1 REGULATES CELL CYCLE IN CANCER CELLS

Figure 2. hnRNP A1 is required for the growth of oral squamous carcinoma cells. A: 5  105 CAL 27 or T3 cells were seeded into a six well plates and transfected by 10 nM hnRNP A1 siRNAs or NS siRNA in the presence of lipofectamine 2000 on Day 0. Cells were passed and received by another transfection on Day 2. The number of cells was counted on Day 2 and 4. Western blotting showed the knockdown efficiency of hnRNP A1. b-actin served as the loading control. A1/b-actin represents the normalized A1 expression levels. B: 2  105 293 cells were seeded into a 12 well plate and transfected with 0.5 mg T7 tagged hnRNP A1 expression or empty vector plasmid in the presence of Lipofectamine 2000 on Day 0. Cells were passed and received by another transfection on Day 2. The number of cells was counted on Day 2 and 4. Western blot analysis showed endogenous and overexpressed exogenous T7 tagged hnRNP A1 with an anti-hnRNP A1 monoclonal antibody. *P < 0.05.

GCCCAGGCTCCAGATGTCCAC-30 (exon 5) for full length CDK2. The relative expression level of each gene was calculated by 2DDCT method. For alternative splicing of CDK2, the ratios of relative expression level of short versus long isoform was calculated using the formula 2DCT, where DCT ¼ (CT short isoformCT long isoform). The data presented here are the average of at least three independent experiments.

sequences of A1 target genes verified by RT-PCR and qRT-PCR were submitted to MEME analysis. For CDK2, the sequence including exon 5 and nearby intron (upstream and downstream 200 nt) was further analyzed by SFmap (an online program for motif analysis and prediction of splicing factors, http://sfmap.technion.ac.il/index.html) (Paz et al., 2010). In addition, we manually searched for a known A1 binding site, UAGGGA/U, in the mRNA sequences of target genes. Statistical analysis

Binding site analysis

A1 binding site analysis was performed using multiple Em for motif elicitation (MEME) online service (Bailey et al., 2009) (http://meme.nbcr.net/meme/cgi-bin/meme.cgi). The cDNA JOURNAL OF CELLULAR PHYSIOLOGY

The scores of hnRNP A1 in tissue array were compared between groups using the nonparametric Mann–Whitney U-test (normal vs. tumor samples) or Kruskal–Wallis test (different grades) in SPSS software. For cell growth, cell cycle,

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Figure 3. hnRNP A1 is associated with the G2/M phase of cell cycle. (A–C) CAL 27 cells in different cell cycle phases were sorted in Beckman Coulter MoFloTM XDP cell sorter. Western blot (A), and RT-PCR (B) were performed to examine the expression of hnRNP A1 in different phases at protein or RNA level. b-actin or GAPDH served as the loading control. A qRT-PCR was also performed to analyze the relative hnRNP A1 mRNA levels (normalized by actin or GAPDH) in different cell cycle stages. Panel C shows the ratios of G2/M to G0/G1, or to S, respectively. (D,E) Cell cycle analysis of CAL 27 (D) and T3 (E) cells. Cells were transfected twice as in Figure 2. Experiments were performed twice and analyzed by Modfit LT software and drawn as column charts by EXCEL. The right-most panels show the summary and statistical analysis of two independent experiments.

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and real-time RT-PCR, two-group statistical comparisons of means were calculated with student’s t-test using Excel (Microsoft).

in both CAL 27 and T3 primary cancer cells induced prominent G2/M cell cycle arrest (Fig. 3D–E), suggesting that hnRNP A1 is associated for G2/M transition.

Results Expression of hnRNP A1 in OSCC.

Target genes of hnRNP A1 in cell cycle G2/M phase

We took advantage of a tissue array (including 49 OSCC tumor samples and 10 normal oral mucosal samples) to analyze the expression of hnRNP A1 in human OSCCs. Immunohistochemistry staining results showed that hnRNP A1 was significantly highly expressed in OSCC samples compared with normal control samples (Fig. 1A). Moreover, we calculated expression scores based on staining intensity and the percentage of cells stained. The expression scores of the OSCC samples were significantly higher than the normal control samples (P ¼ 0.006, Fig. 1B). However, expression scores of OSCC were not associated with the grades of OSCC samples (P ¼ 0.956, Fig. 1C). We also analyzed the expression of hnRNP A1 in cultured tumor and normal cells. CAL 27 (an OSCC cell line) and three primary cultured OSCC tumor cells showed significantly higher levels of hnRNP A1 than four normal oral mucosal primary cultured cells (Fig. 1D). These results suggest that hnRNP A1 may be an important factor involved in most OSCC cases, no matter how malignant. hnRNP A1 is required for the growth of OSCC cells

Next, we analyzed the function of hnRNP A1 in OSCC. Knockdown of hnRNP A1 in either CAL 27 or primary oral cancer cell significantly reduced cell proliferation compared with NS siRNA (Fig. 2A), indicating that hnRNP A1 is required for the growth of OSCC cells. Two hnRNP A1 siRNAs showed similar results, suggesting that the cell growth retardation may not be due to the off-target effects. Moreover, overexpression of hnRNP A1 in 293 cells significantly promoted cell growth (Fig. 2B), which confirmed that hnRNP A1 is important for cell growth.

To further understand the mechanism of the effect of hnRNP A1 on G2/M transition in OSCC cells, we compared gene expression profile in hnRNP A1 or NS siRNA treated cells by DNA microarray assay. Twelve genes with significant changes between two groups (P < 0.05, at least 1.5-fold change) and involved in G2/M phase were identified in microarray analysis, including SEPT8, CDK2, CLASP1, DNM2, IGF2 (Fernandez-L et al., 2012), NEK9, NEK11, NRAS (Pomp et al., 1996), PCTK1, PARD3B (Hong et al., 2010), RCC2, and SEPT3 (Table 1). Eight of these genes were validated by RT-PCR and qRT-PCR (Figs. 4 and 5). Importantly, alternative splicing of exon 5 of CDK2, one of the targets, was regulated by hnRNP A1. Based on Genbank database, human CDK2 has at least two isoforms generated by alternative splicing of exon 5 (102 nt). Isoform 2 has 34 aa inframe deletion compared with isoform 1. Both RT-PCR and qRT-PCR showed that knockdown of hnRNP A1 decreased the levels of isoform 1(Fig. 5B,C). Western blotting also confirmed the decreased expression of CDK2, although no isoform 2 was detectable in all samples (Fig. 5D). In addition, we analyzed the expression of both hnRNP A1 and CDK2 in the same western blot membrane (Fig. S2). The result showed that the expression of CDK2 correlated with hnRNP A1 in primary human oral squamous cancer cells, CAL 27 cells, or normal primary oral mocosal epithelial cells (Fig. S2). However, the difference between two isoforms of CDK2 remains unknown. We discovered that overexpression of isoform 1 promoted cell growth, whereas overexpression of isoform 2 relatively inhibited cell growth in both 293 and CAL 27 cells (Fig. 5E–H), suggesting that hnRNP A1 may affect cell proliferation through the regulation of alternative splicing of CDK2 exon 5. Taken together, these results suggested that hnRNP A1 may control cell cycle transition by regulating the expression and alternative splicing of a number of genes associated with cell cycle G2/M phase.

hnRNP A1 is associated with G2/M stage

Dysregulation of cell cycle is the key event in oral carcinogenesis (Todd et al., 2002). Thus, we analyzed the expression and function of hnRNP A1 in cell cycle. Cells in different cell cycle stages were sorted and analyzed by western blotting (Fig. 3A), RT-PCR (Fig. 3B), and qRT-PCR (Fig. 3C). Notably, western blotting, RT-PCR, and qRT-PCR results showed that hnRNP A1 was highly expressed in G2/M stage in CAL 27 cells compared with G0/G1 and S stage. Knockdown of hnRNP A1

hnRNP A1 binding sites

MEME analysis of target mRNA sequences showed a most consensus sequence: G(G/C)AG(C/G)AG(C/A/G) (Figs. S3A, B). SFmap analysis of CDK2 exon 5 and nearby intron showed that an hnRNP A1 binding motif, GUAGUAGU, is located in intron 4. Searching for a known A1 binding site in target mRNA sequences showed that six target genes contain 10 “UAGGA/ U” sites.

TABLE 1. List of G2/M related genes regulated by A1. CAL 27 cells were treated with hnRNP A1 or NS siRNA twice in a 48-hour interval. Total RNA was used in a DNA microarray assay. Differentially expressed genes with statistical significance were identified. The table shows 12 G2/M-related genes with significant changes between two groups (P < 0.05, at least 1.5 fold change) Gene name 1 2 3 4 5 6 7 8 9 10

CDK2 CLASP1 DNM2 IGF2 NEK11 NEK9 NRAS PCTK1 PARD3B RCC2

11 SEPT3 12 SEPT8

Description

Fold NCBI P-value change Regulation Gene ID

Cell cycle

Reference (Rosenblatt et al., 1992) (Maiato et al., 2003a, 2003b) (Ishida et al., 2011) (Fernandez-L et al., 2012) (Melixetian et al., 2009) (Kaneta and Ullrich, 2013) (Pomp et al., 1996) (Charrasse et al., 1999) (Hong et al., 2010), (Mollinari et al., 2003; Grigera et al., 2012) (Trimble, 1999) (Trimble, 1999)

Cyclin-dependent kinase 2 Cytoplasmic linker associated protein 1 Dynamin 2 insulin-like growth factor 2 (somatomedin A) NIMA (never in mitosis gene a)- related kinase 11 NIMA (never in mitosis gene a)- related kinase 9 Neuroblastoma RAS viral (v-ras) oncogene homolog PCTAIRE protein kinase 1 (CDK16) Amyotrophic lateral sclerosis 2 Regulator of chromosome condensation 2

0.0004 0.0006 0.0025 0.0093 0.0221 0.0198 0.0022 0.0057 0.0308 0.0151

1.66 1.72 1.70 1.80 1.80 1.56 1.56 1.54 1.79 1.50

Down Down Down Down Down Down Down Down Up Down

1017 23332 1785 3481 79858 91754 4893 5127 117583 55920

S/G2 phase M phase G2/M phase G2/M phase S, G2/M2 phase M phase G2/M phase S/G2 phase M phase G2/M phase

Septin 3 Septin 8

0.0392 0.0016

1.95 1.61

Down Down

55964 23176

M phase M phase

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Figure 4. hnRNP A1 regulates the expression of G2/M related genes. (A,B) Total RNA was collected from CAL 27 cells transfected by siRNA twice in Figure 2. Microarray data analysis showed the expression of 12 G2/M-related genes was significantly changed after hnRNP A1 knockdown. Seven of these target genes were verified by RT-PCR (A) or qRT-PCR (B,C), namely, CLASP1, NEK11, NEK9, NRAS, RCC2, SEPT3, and SEPT8. A: GAPDH served as the loading control. The numbers below indicated relative expression level of target genes in hnRNP A1 silenced cells compared with NS siRNA treated cells. (B,C) Relative expression levels of A1 target genes were normalized by GADPH (B) or actin (C) expression levels. *P < 0.05.

Discussion

hnRNP A1 is a multifunctional protein known to be involved in human diseases including cancers. Considerable studies have reported the overexpression of hnRNP A1 in various cancers JOURNAL OF CELLULAR PHYSIOLOGY

(Jean-Philippe et al., 2013). Consistent with these studies, we also discovered that hnRNP A1 is overexpressed in OSCC tissues or cultured cells compared with normal oral mucosal tissues or cultured primary epithelial cells, indicating that hnRNP A1 may be associated with OSCC. Few study analyzed

hnRNP A1 REGULATES CELL CYCLE IN CANCER CELLS

Figure 5. hnRNP A1 regulates alternative splicing of CDK2. A: Exon 5 of CDK2 undergoes alternative splicing. Inclusion or exclusion of exon 5 produces isoform 1 or 2. (B,C) Alternative splicing of CDK2 exon 5 is regulated by hnRNP A1. Total RNA was from CAL 27 cells transfected by hnRNP A1 siRNAs in Figure 2. RT-PCR (B) and qRT-PCR (C) were used to analyze alternative splicing of CDK2 exon 5 in CAL 27 cells treated with hnRNP A1 or non-specific (NS) siRNA (B). S/L: the ratio of short product versus long product. GAPDH served as the loading control. D: Western blot analysis of CDK2 expression in CAL 27 cells without treatment or treated with hnRNP A1 or NS siRNA. b-actin served as the loading control. (E,F) 293 (E) or CAL 27 (F) cells were transfected twice with GFP tagged CDK2 isoform 1 or 2 expression plasmid in a 48-hour interval. Cells transfected with pEGFP-N1 plasmid served as a control. The number of cells was counted on 96- or 72hour after first transfection, respectively. (G–H) Western blot analysis of GFP tagged CDK2 expression in 293 (G) or CAL 27 (H) cells with mouse anti-CDK2 or anti-GFP monoclonal antibody.

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the correlation between expression of hnRNP A1 and tumor progression. We have found that expression of hnRNP A1 is independent of OSCC grade, suggesting that overexpression of hnRNP A1 may be an early pathogenic event in OSCC. Previous studies have indicated that hnRNP B1 (Goto et al., 1999) has the potential to be the useful marker for early detection of OSCC. hnRNP A1 has potential as new biomarker for OSCC. hnRNP A1 has multiple isoforms generated from alternative splicing and posttranslational modification (Carpenter et al., 2006). Alternative splicing produces isoform 1 (NM_002136.2, 34 kD) and isoform 2 (NM_031157.2, 38 kD) (Buvoli et al., 1990). Using a mouse monoclonal anti-hnRNP A1 antibody, we can detect two bands by long exposure in western blotting. The major band is slightly bigger than 38 kDa, while another weak band is smaller than 38 kDa (Fig. S1), indicating that isoform 2 of A1 is the major isoform expressed in OSCC and oral mucosal epithelial cells. However, the functional differences between these two isoforms have not been distinguished. We found that hnRNP A1 is essential for proliferation of oral squamous cell carcinoma. However, reports about the function of hnRNP A1 in cell proliferation are contradictory. Li et al. (2012) found that downregulation of hnRNP A1 inhibited proliferation of HepG2 cells (hepatocellular carcinoma cells). He et al. (2005) showed that cell proliferation of Colo16, a skin squamous cell carcinoma, was affected by knocking down both of hnRNP A1 and A3 instead of individual gene knockdown. Ben-David et al. (1992) found that a mouse erythroleukemia cell line grew well without hnRNP A1. Yeh et al. (2014) reported that knockdown of hnRNP A1 or A3 did not affect Jurkat cell proliferation. Therefore, the function of hnRNP A1 in cell proliferation may be cell-type dependent. Hematogenous tumor cells seem to be resistant to the depletion of hnRNP A1. A1 may also functionally overlap with other factors in cell proliferation. Notably, Planck et al. (1988) showed that hnRNP A1 mRNA levels remarkably increased after the stimulation of serum or epidermal growth factor. More analysis on different cell types may be required to clearly address this concern. In the present study, we found that hnRNP A1 was required for cell proliferation, and proved that A1 was associated with G2/M cell cycle phase. We discovered that A1 was highly expressed in G2/M cell cycle phase. Depletion of A1 caused G2/M arrest. Levels of a number of G2/M related genes had been regulated by A1 (Table 1). Most of these proteins are important for the progress of mitosis, such as SEPT3, SEPT8 (Trimble, 1999), CLASP1 (Maiato et al., 2003a, 2003b), RCC2 (Mollinari et al., 2003; Grigera et al., 2012), NEK9 (Kaneta and Ullrich, 2013), DNM2 (Ishida et al., 2011), and NEK11 (Melixetian et al., 2009). These proteins are associated with the control of cell cytoskeleton assemble or arrangement during mitosis. Expressions of two cyclin-dependent kinases, namely, CDK2 and CDK16 (Charrasse et al., 1999), are also controlled by hnRNP A1. CDK2 complexes with cyclin E are essential to drive the G1/S transition. CDK2 can also be activated by cyclin A2 in G2 phase (Rosenblatt et al., 1992). Although CDK2 has been shown to be unimportant for most cell proliferation and no effect on cell cycle in 293T cells (Breuer et al., 2012), it is required for meiotic division of both male and female germ cells (Ortega et al., 2003). Moreover, CDK2 is essential for the survival of tumor cells with N-MYC overexpression (Molenaar et al., 2009). Amino acids from Val163 to Met-196 are deleted in CDK2 isoform 2. This region is located in the middle of protein kinase domain and may affect CDK2 functions. To the best of our knowledge, the functions of these two isoforms have been undefined. We discovered that overexpression of CDK2 isoform 1 promoted cell growth, whereas overexpression of isoform 2 did not. JOURNAL OF CELLULAR PHYSIOLOGY

hnRNP A1 binds to specific sequences of pre-mRNA. Burd and Dreyfuss (1994) found that A1 has high affinity to a sequence, namely, UAGGGA/U. The interaction between this sequence and A1 is so significant that it can be used as a specific affinity reagent for A1. We also found this sequence in target genes. This phenomenon suggests that A1 may directly bind to these targets. Analysis of CDK2 alternative exon 5 and nearby intron sequence showed that an additional hnRNP A1 binding motif, namely, GUAGUGUU (Akerman et al., 2009), is located in intron 4. hnRNP A1 may bind to this site and regulate the alternative splicing of CDK2 exon 5. Moreover, we predicted a possible A1 binding site, namely, G(G/C)AG(C/G)AG(C/A/G), based on target mRNA sequences. Further experiments are required to confirm this binding site. Overall, our results demonstrated that hnRNP A1 has important roles in cell proliferation by regulating a series of genes involved in G2/M phase. Overexpression of hnRNP A1 may be a potential biomarker of OSCC. Acknowledgments

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