Aptamer BC15 Against Heterogeneous Nuclear Ribonucleoprotein A1 Has Potential Value in Diagnosis and Therapy of Hepatocarcinoma

Abstract

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was reported to be participated in tumor development. The association between hnRNP A1 and liver cancer and the functional role of hnRNP A1 in liver cancer have never been reported. Herein, hnRNP A1-specific single-stranded DNA aptamer, BC15, was used to (a) evaluate hnRNP A1 expression in liver cancer, and (b) treat hepatocarcinoma by acting as an inhibitor of hnRNP A1. Results showed that there is high hnRNP A1 expression in liver cancer including serum α-fetoprotein–negative liver cancer tissues compared with either para-cancer or benign controls. Down regulation of hnRNP A1 expression by RNA interference inhibits the proliferation and migration of cancerous HepG2 cells, while overexpression of hnRNP A1 in normal HL-7702 cells increased the proliferation and migration of the cells. Importantly, BC15 showed a stronger inhibiting effect on the proliferation of cultured hepatoma cells than hnRNP A1 small interfering RNA, strongly suggesting that BC15 could also be a potential drug candidate for an hnRNP A1 inhibitor besides its prospect utility in in situ histological examination.

Introduction

Primary carcinoma of the liver, including hepatocellular carcinoma (HCC) and intra-hepatic cholangiocarcinoma, is the third most common cause of cancer-related death, with more than 500,000 new cases diagnosed annually worldwide (RAOUL, 2008). Alpha-fetoprotein (AFP), the first tumor marker identified for this disease, plays an important role in the diagnosis and treatment of liver cancers. Because 30%–40% of liver cancers are AFP-negative (Yao et al., 2000), novel biomarkers and the corresponding probes for these cancers are urgently needed, not only for diagnosis but also for targeted biological therapy (Santamaría et al., 2007).

Aptamers evolved from SELEX (systematic evolution of ligands by exponential enrichment) is a class of concerned biomedicine with high specific affinities for a variety of targets such as metal ions, organic dyes, and proteins. The recently developed cell-based SELEX (Wang et al., 2003; Shangguan et al., 2006) and tissue slides-based SELEX (Li et al., 2009) techniques provide convenient ways to identify aptamers distinguishing cancer cells from clinical samples even without knowing the molecular differences beforehand. The developed aptamers were further used to identify the molecular differences (i.e., the biomarkers) and used as a novel and powerful class of probes for diagnostic and therapeutic applications due to their superiority to antibodies such as easy synthesis, less immunogenicity, lower molecular weight, and high specificity. The newly developed structural DNA nanotechnology even makes aptamers a hot topic in nucleic acid biomedicine and cell-targeting task (Douglas et al., 2012; Sundaram et al., 2012).

In a previous study, we obtained a single-stranded DNA aptamer, BC15, which specifically differentiates breast cancerous tissues from normal ones through recognizing heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) (Li et al., 2009). Moreover, work from this group has also shown that BC15 can recognize hnRNP A1 in a variety of cancerous tissues (Li S, Ding H, Xu H et al. Beijing Institute of Basic Medical Sciences. unpublished data), consistent with reports that hnRNP A1 overexpression was observed in breast, lung, ovary, and colon-derived cancers (Patry et al., 2003; Ushigome et al., 2005; Ma et al., 2009; Boukakis et al., 2010). As hnRNP A1 expression in liver cancer has not been reported yet, we evaluated the role of the pair of molecules (BC15 and hnRNP A1) in liver cancer in this study, where BC15 may have utility in the diagnosis and treatment of liver cancer.

Materials and Methods

Clinical samples and cell lines

Tissue samples were obtained from 73 patients admitted for liver surgery who had serum AFP levels detected and confirmed diagnosis given at the General Hospital of Fuzhou, China. The excised tissue samples were fixed in formalin and serially sectioned (4 μm). One of the sections was then stained for routine hematoxylin and eosin histopathology, and the sections containing regions of interest were categorized as either being cancerous or benign, and adjacent areas with no evidence of cytomorphological changes were used as normal controls. With respect to the cell culture, the hepatoma cell line HepG2, SMMC-7721, and normal liver cell line HL-7702 (Hou., et al. 2008) were provided by Shanghai Cell Bank of Chinese Academy of Sciences and cultured in minimum essential medium (MEM) or Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 10% or 20% fetal bovine serum (Gibco, New York) respectively. The HepG2 cell line stably expressing enhanced green fluorescent protein (HepG2-EGFP) was established commercially (Genechem Co., Shanghai, China).

Identification of hnRNP A1 expression and its relationship to mutant p53 expression

The BC15 (5′-GCAATGGTACGGTACTTCCTGTGGCGAGGTAGGTGGGGTGTGTGTGTATCCAAAAGTGCACGCTACTTTGCTAA-3′) and control (GP30: 5′-GCAATGGTACGGTACTTCC(N30)CAAAAGTGCACGCTACTTTGCTAA-3′) were synthesized and 5′ labeled with FAM (6-carboxyfluorescein, Shangon Co. Ltd. Shanghai, China). The paraffin-embedded slides were deparaffinized in xylene, rehydrated using graded ethanol, dipped into 0.01 M sodium citrate buffer, and heated in a microwave oven at 96–98 °C for 10 minutes (antigen retrieval) before the experiment. The expression of hnRNP A1 was then assessed using FAM-labeled BC15 or using a primary anti-hnRNP A1 antibody (AVIVA Systems Biology, Co, Beijing, China) and fluorescein isothiocyanate–labeled secondary antibody as previously described (Li et al., 2009). Evans' blue was used to reduce the background of nonspecific auto fluorescence and to help distinguish the specific green fluorescence labeling from the red background. To assess the expression of hnRNP A1 relative to mutant p53 expression in the same tissue slides, the slides were first probed with mouse anti-mutant p53 monoclonal antibody and tetramethyl-rhodamine-5(and-6)-isothiocyanate (TRITC)-labeled secondary antibody, and then probed with FAM-labeled BC15. The pattern of expression was then evaluated using a Zeiss LSM 510META confocal microscope or converted fluorescence microscope (Olympus IX71). Staining was compared to the negative control (−) and categorized as follows: (+)=<25% of cells stained; (++)=26%–50% of cells stained; (+++)=51%–75% of cells stained; and (++++)=>75% of cells stained.

RNA interference assay

The cells were plated into 6-well plates (3×10⁵ per well). After 24 hours, the small interfering RNA (siRNA; 100 pmol/well) was transfected into the cells using Lipofectamine 2000 (5 (L/well). After 48 or 72 hours the cells were collected and used for the experiments. The siRNA-specific for hnRNP A1 (siRNPA1: 5′-CUUUGGUGGUGGUCGUGGATT-3′, 3′-TTGAAACCACCACCAGCACCU-5′) and the control siRNA (NC: 5′-UUCUCCGAACGUGUCACGUTT-3′, 3′-TTAAGAGGCUUGCACAGUGCA-5′) were synthesized chemically (Gene Pharma, Shanghai, China).

Stable overexpression of hnRNP A1 in the HL-7702 cell line

The hnRNP A1 gene was amplified by polymerase chain reaction (PCR) using complementary (cDNA) library of HepG2 as the template and primers (hnRNP A1-F1-Bgl2, 5′-GAAGATCTATGTCTAAGTCAGAGTCTCC-3′ and hnRNP A1-R1-Hpa1, 5′-ACTTGTTAACTTAAAATCTTCTGCCACTGC-3′, Invitrogen Co., Ltd., Beijing, China). The sequence was verified to be correct by sequencing PCR product (Shangon Co., Shanghai, China). Then the PCR product was cloned into Bgl2 and Hpa2 restriction sites of the retroviral vector pMSCVpuro through DNA recombination to generate pMSCV-hnRNP A1. The recombinant plasmid was transformed into competent DH5α, and the positive clones were picked up for sequencing and propagating using plasmid purifying kit (QIAGEN, Co., Hilden, Germany). The 293T packaging cells were plated on a 60-mm plate at a density of 1–2×10⁶ cells and allowed to adhere overnight in a 5% CO₂ incubator at 37°C. The cells were rinsed with medium and then transfected with 20 μg of the recombinant vector by standard calcium phosphate method, and incubated for 6 hours at 37°C. Fetal bovine serum (10%) was subsequently added to the medium and cells were incubated for 24 hours. Viral supernatant was recovered, filtered through a 0.45-μm cellulose acetate filter, and added to HL-7702 cells plated at 60% confluence. The polybrane was added to the medium to a final concentration of 4 μg/mL to increase the infection efficiency. At successive 24-hour intervals for the following 3 days, medium from transfected 293T cells was added onto HL-7702 cells. Infected HL-7702 cells were cultured for 24 hours before selected with puromycin (3 μg/mL).

Total protein extraction and immunoblotting

Total protein was extracted using radio immuno-precipitation assay (RIPA) lysis buffer (150 mM sodium chloride, 50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.1% SDS) in the presence of a proteinase inhibitor cocktail (Sigma-Aldrich, Saint Louis, MO). The immunoblotting was performed as previously described (Li et al., 2008) using primary an anti-hnRNP A1 polyclonal antibody (AVIVA Systems Biology, Co, Beijing, China).

Cell proliferation assay

Hepatoma HepG2 and SMMC-7721 cells transfected with siRNPA1 (10 pmol/well), BC15 (10 pmol/well) or controls were seeded into 96-well culture plates (2×10³ per well; NUNC, Roskilde, Denmark), and then incubated in MEM supplemented with 10% fetal bovine serum (Gibco) at 37°C/5% CO₂ for 3–4 days. Cell proliferation was assessed by adding 0.5 mg/mL MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; thiazolyl blue). After a 4-hour incubation period, the medium was discarded and 0.1 mL dimethyl sulfoxide (DMSO) was added to each well for 10 minutes at room temperature. Cells viability was then evaluated by measuring absorbance at 595nm. Changes in cell proliferation were similarly evaluated using HL-7702 cells that stably overexpressed hnRNP A1 (A1), or were infected with pMSCVpuro (P) and blanks (Mock).

Transwell cell migration assays

For the Boyden chamber assay, the cells were serum starved for 24 hours, washed twice with phosphate-buffered saline, and resuspended in serum-free medium. A 200-(L sample of the migratory cell mix (2×10⁵ per mL) was added to the transwell inserts, and 600 μL of medium supplemented with 10% fetal bovine serum was added to the bottom chamber. The cells were allowed to migrate for 8 hours (HepG2) or 16 hours (HL-7702) at 37 °C. Unmigrated cells were removed from the inner chamber/top of the membrane using a cotton swab. The cells were fixed with methanol for 20 minutes at −20°C, stained with Giemsa or crystal violet, and evaluated.

Liver cancer animal model

Cohorts of 6- to 8-week-old nude mice were purchased from the Beijing Laboratory Animal Centre, Beijing, China. The mice were randomly divided into 3 groups (n≥15 per group), and injected subcutaneously at a single site with 1×10⁶ HepG2-EGFP cells transiently transfected with BC15 (2 (g, about 67 pmol) or siRNA1 (60 pmol), or with HepG2-EGFP cells treated with transfection regents (in vivo-jetPEI™, Polyplus-transfection, Illkirch, France). All animal experiments were undertaken in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. For 14 days, the 3 groups of mice were observed for the presence of tumor formation, at which point the BC15 aptamer (0.7 μg/mouse, about 23 pmol/mouse), siRNA1 (20 pmol/mouse), or transfection regents were percutaneously injected into the tumors 3 times every other day for the next 6 days. Finally, the mice were sacrificed and the tumors were harvested and weighed.

Statistical analysis

Each experiment was performed at least twice. The statistical significance of the results was determined using SPSS v19.0 software through Student's t-test or chi-square test. A value of P<0.05 was considered statistically significant.

Results

BC15 probing revealed high hnRNP A1 expression in primary liver carcinomas

Because BC15 had been proven to recognize hnRNP A1 specifically, we used FAM-labeled BC15 to evaluate hnRNP A1 expression in a set of samples obtained from 73 patients admitted for liver surgery. Of these patients, hospitalization data revealed 54 cases of HCC, 8 cases of cholangiocarcinoma, and 11 cases of benign hepatic lesions or normal controls. Results revealed that 71% of the tissues obtained from patients with primary carcinoma of the liver were hnRNP A1-positive compared with 18.2% of those from patients with benign liver disease (Table 1). Of the 62 cases where cancer was detected, 32 of these cases were characterized by low serum AFP levels (<200 ng/mL). Further, for these patients the frequency of positive expression hnRNP A1 was 75% (Table 1). This suggests that using BC15 to evaluate hnRNP A1 expression may be of use clinically as a potential adjunct in situ biomarker for the diagnosis of liver cancer.

Table 1.

Expression of hnRNP A1 in Primary Liver Carcinomas

		Numbers (%)
Samples		++++	+++	++	+	–	Total positive
Pathological characters	HCC	7 (13.0)	12 (22.2)	6 (11.1)	12 (22.2)	17 (31.5)	37 (68.5)
	Cholangiocarcinoma	3 (37.5)	4 (50.0)	0	0	1 (12.5)	7 (87.5)
Serum AFP	Cancer with serum AFP <200 ng/mL	4 (12.5)	7 (21.9)	4 (12.5)	9 (28.1)	8 (25.0)	24 (75.0)
	Cancer with serum AFP ≥200 ng/mL	6 (20.0)	9 (30.0)	2 (6.67)	3 (10.0)	10 (33.3)	20 (66.7)
Total liver cancer		10 (16.1)	16 (25.8)	6 (9.7)	12 (19.4)	18 (29.0)	44 (71.0)
Benign/normal		0	1 (9.1)	0	1 (9.1)	9 (81.8)	2 (18.2)

n=73.

AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma.

Association between BC15 binding intensity and other clinicopathologic parameters in liver cancer

It was also revealed that hnRNP A1 expression appeared to be correlated with the various levels of HCC differentiation, from well differentiated to poorly differentiated (Fig. 1A), indicating that hnRNP A1 may be involved in the tumor development process. We also investigated the relationship between BC15 binding intensity and different clinicopathologic parameters. Of the 53 cases where diagnostic imaging data was available, a higher frequency of BC15 positive binding in the interlobular portion of the liver was observed in patients accompanied by tumor thrombosis of the portal vein or tumor invasion into other tissues (9/15, 60%) than in the patients with noninvasive tumors (10/38, 26%; P<0.05) (Table 2, Fig. 1B). Moreover, hnRNP A1 expression was positively correlated with the mutant p53 expression (P<0.05 for Spearman's correlation) (Table 3, Fig. 1C), but no relationship was noticed between hnRNP A1 and Ki-67, P-glycoprotein, DNA-topoisomerase II, EGFR, or vascular endothelia growth factor (VEGF) protein levels (P>0.1 for Spearman's correlation) (Table 3).

FIG. 1.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) expression is linked to prognostic factors in hepatocellular carcinoma (HCC) samples. (A) hnRNP A1 expression detected by 6-carboxyfluorescein-labeled hnRNP A1-specific single-stranded DNA aptamer (FAM-BC15) is linked to cell differentiation. Scale bar=20 (m. (B) Representative images showing hnRNP A1 expression in the interlobular portion, or zones of the portal tract from liver carcinoma samples evaluated using FAM-labeled BC15. In each case, the right panel is the magnification of the left one. Scale bar=20 (m. (C) Representative images showing hnRNP A1 expression is related to mutant p53 expression. Three cases of hnRNP A1 of high, moderate, and negative expression with a comparable level of mutant p53 are shown.

Table 2.

Relationship Between Expression of hnRNP A1 in the Interlobular Portion, or Zones of the Portal Tract and Tumor Surrounding Invasion

	hnRNP A1 expression numbers (%)
Invasion	++++	+++	++	+	–	Total positive
Yes	1 (6.7)	5 (33.3)	2 (13.3)	1 (6.7)	6 (40.0)	9 (60.0)
No	1 (2.6)	4 (10.5)	3 (7.9)	2 (5.3)	28 (73.7)	10 (26.3)

n=53.

Table 3.

Pathologic Significance of hnRNP A1 Overexpression Detected by BC15 in Tissues from Primary Carcinoma of the Liver

		BC15 reactive intensity
Variables	No.	Negative	Low	High	Value (P)
Mutant p53					10.031 (0.04)^*, 0.474(0.006)†
Negative	9	6	1	2
Low	12	2	4	6
High	11	1	2	8
Ki-67 index					6.362 (0.042)^*, 0.171(0.349)†
<50	22	6	7	9
≥50	10	3	0	7
P-gp					1.977 (0.372)^*
Low	4	1	2	1
High	28	8	5	15
TOPOII					4.118 (0.390)^*
Negative	2	1	0	1
Low	28	8	7	13
High	2	0	0	2
EGFR					7.686 (0.104)^*
Negative	4	3	1	0
Low	7	2	2	3
High	21	4	4	13
VEGF					2.065 (0.724)^*
Negative	4	1	2	1
Low	10	3	2	5
High	18	5	3	10

Likelihood ratio chi-square test; †Spearman's correlation test.

n=32 cases open to hospital pathological inspections including mutant p53, Ki67, P-glycoprotein (P-gp), DNA-topoisomerase II alpha (TOPOII), epidermal growth factor receptor (EGFR), and vascular endothelia growth factor (VEGF) expression by traditional immunohistochemical staining. All the primary antibodies were purchased from Maixin Biology, Co., Fuzhou, China. The binding signal was developed with ultrasensitive S-P kit in accordance with the manufacturer's instructions. We defined that less than 50% represented low, more than 50% represented high, and less than 5% represented negative mutant p53, p-gp, TOPOII, EGFR, and VEGF expression. The Ki-67 labeling index was calculated as the percentage of immunopositive of cells.

The role of hnRNP A1 in the hepatoma proliferation and migration in vitro

Given the higher expression level of hnRNP A1 in the cancerous tissues relative to the normal or benign tissues, as well as the correlation between hnRNP A1 expression and mutant p53 levels and its correlation with structural invasion, the role of hnRNP A1 in hepatocellular carcinoma was tested in HepG2 and SMMC-7721 cells using knockdown assays and using overexpression experiments in the HL-7702 cells. In a previous study, we verified that siRNPA1 can specifically downregulate hnRNP A1 expression level (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/nat). Therefore, we silenced hnRNP A1 in HepG2 cells using siRNPA1 and showed inhibited proliferation and migration of HepG2 cells in vitro (Fig. 2A), while hnRNP A1 overexpression in the HL-7702 cells increased their proliferation and the migration of the cells in vitro (Fig. 2B). Results also showed that BC15 had a stronger and longer-lasting growth inhibiting effect than siRNPA1, as student's t-test suggested that BC15 showed significant growth inhibition effect at 36 hours, 48 hours, and 60 hours after transfection to HepG2 cells, while siRNPA1 showed a significant growth inhibition only at 36 hours and 48 hours after transfection. We reproduced the results in SMMC-7721 cells, where BC15 showed a significant inhibition from 24 hours to 84 hours at sample points, and siRNP A1 showed effect only from 24 hours to 60 hours at sample points (Fig. 2A).

FIG. 2.

The effect of hnRNP A1 on the proliferation and migration of hepatoma cell line (HepG2) or normal liver cell line (HL-7702) cells in vitro. (A) RNA interference assays in HepG2 cells. (B) Overexpression experiments in HL-7702 cells. (a) Immunoblots showing hnRNP A1 protein level. (b) MTT assays to evaluate the proliferation changes of cells caused by treatment. Cells were transfected, seeded in 96-well microplates in quadruplicate, and cultured for up to 4 days. Cell proliferation at different times after transfection was assessed by the MTT assay. Each experiment was repeated in triplicate. The results are shown as mean±standard deviation. Left, HepG2 cell line. Right, SMMC-7721 cell line. (c) Transwell cell migration assays. The alterations in migration by hnRNP A1 treatment were assessed by the Boyden chamber assay. The migrated cells on the bottom of the membrane were fixed with methanol, stained with Giemsa or crystal violet, photographed, and evaluated by cell counting. **P<0.01 (compared with the NC negative control or plasmid control). A1, cells with hnRNP A1 overexpression; Mock, blanks; P, cells transfected with the retroviral vector pMSCVpuro.

BC15 inhibit tumor growth in a HepG2 xenograft model

In order to evaluate whether hnRNP A1 may be a useful therapeutic target and explore the potential of BC15, we tested whether manipulating hnRNP A1 affected tumor growth in vivo using a HepG2-EGFP xenograft model. These experiments revealed that siRNPA1 and BC15 treatment resulted in a tendency of tumor growth inhibition as compared with the transfection regent control (Fig. 3). The suppression between BC15 and aptamer control GP30 as well as that between siRNA and NC were of no significance (P>0.05), which may be due to the small number of experimental subjects, wide individual variation, poor transfection efficiency in solid tumors, insufficient nucleic acids therapeutics after quick metabolism or degradation in vivo, and importantly, off-target effect of the controls in vivo especially GP30 is a library of about 10¹⁸ sequences (4³⁰ sequences). It also appeared that BC15 conferred a protective effect in the xenograft model, with a lowered death rate (1/24, 4.167%) than siRNA (3/21, 14.286%), GP30 (6/22, 22.273%), and transfection regents groups (6/15, 40%).

FIG. 3.

The effect of hnRNP A1 siRNA or BC15 on tumor growth. The HepG2-epidermal growth factor receptor (EGFP) cells transfected with siRNA, BC15 or controls were injected into the nude mice. After 14 days of tumor formation and 3 subsequent doses nucleic acids (administered every other day) injected percutaneously into the tumors, the tumors were harvested and weighed. Weight is shown as a mean±standard deviation. *P<0.05, **P<0.01 (compared with the transfection regent treated group).

Discussion

Aptamers have been promoted as ideal diagnostic reagents and potential antibody alternatives due to the advantages of easy of synthesis and direct labeling (for review, see Soontornworajit and WANG, 2011; Hong et al., 2011). This is the first report in which we use aptamer BC15 to evaluate hnRNP A1 in situ expression in primary liver carcinomas. HnRNP A1 is reported to play important roles in a variety of biological processes (Fiset and Chabot, 2001; Guil and Caceres, 2007; Shi et al., 2008; Paramasivam et al., 2009; Flynn et al., 2011) and tumor development. Recent study reveals that hnRNP A1 was a metastatic indicator in colorectal cancer (He et al., 2010; Hope and Murray, 2011). But association between hnRNP A1 and liver cancer and the functional role of hnRNP A1 in liver cancer have never been reported. In this study we found a consistent high expression of hnRNP A1 in liver cancer. Moreover, hnRNP A1 was also highly expressed in lesions from AFP-negative patients, suggesting that it is a potentially valuable biomarker for the clinical diagnosis of these neoplasms.

We also made the novel discovery that hnRNP A1 expression correlated with mutant p53 levels, and that cancerous cells in the interlobular portion, or in the portal tract, with high hnRNP A1 expression were related to tumor invasion into the surrounding tissues including the portal vein. This finding may allow us to predict the outcome of the tumor after resection biologically. Further experiments showed consistent results that upregulation of hnRNP A1 can promote the proliferation and migration of liver cells in vitro, while knockdown of hnRNP A1 by RNAi and BC15 inhibited the proliferation and the migration of cancerous cells, strongly suggesting that hnRNP A1 is involved in the development and invasion of liver cancer, and thus may be a potential target for biological therapy. However, the mechanisms underlying the relationship between mutant p53 and hnRNP A1 as well as the effect of hnRNP A1 on cell migration are currently unclear and must be verified.

The reports that aptamers were used as target inhibitors or antibody alternatives for the treatment of diseases are increasingly being noted in the literature (for review, see Duncan., 2011; Esposito et al., 2011; Wang P et al., 2011). In this study, BC15 was explored, and the results showed that BC15 can inhibit the proliferation of HepG2 cancer cells and HL-7721 cancer cells. Of note was the discovery that BC15 also showed a stronger inhibitory effect than siRNA. Consistently, BC15 has also been shown to be a more potent inhibitor of proliferation in the breast cancer cell line MCF7 than siRNPA1 (Supplementary Fig. S1). Unfortunately, we did not observe a significant effect of manipulating hnRNP A1 by BC15 or siRNA on tumor growth in the liver carcinoma xenograft mouse model when compared with nucleic acid controls. Because GP30 library and NC control also showed a slight inhibition tendency as they did on growth of HepG2 cell line—therefore, even BC15 and siRNPA1 have significant inhibition effect when compared with the transfection regent—the suppression was of no significance when compared with the nucleic acid control. We deduce that this may be mainly because of off-target effect of the controls in vivo, especially when GP30 is a library of about 10¹⁸ sequences. We did not observe a stronger inhibitory effect of BC15 on tumor growth except for an unexpected, improved survival rate, more so than siRNA in the animal model. This kind of protection effect needs to be studied further. Taken together, these results suggest that BC15 has utility both in evaluating hnRNP A1 expression and in inhibiting hnRNP A1 function. The program of internalizing BC15 with the aid of tumor-specific aptamer or DNA nanostructure technique is under progress and is expected to impact our cancer therapeutics.

Footnotes

Acknowledgments

This work was supported by the Chinese National High-Tech Research and Development Program (2006AA02Z408, SQ2010AA0220970001), the National Natural Science Foundation of China (81000930), and Beijing Scientific Project (Z08050703080809).

We are grateful to all the members of our laboratory for their technical help. We are grateful to Dr. Mengfeng Li and Jun Li (Sun Yat-sen University, Guangzhou, China) for kindly providing us the pMSCVpuro vector.

Author Disclosure Statement

No competing financial interests exist.

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