|
Hepatitis B virus (HBV) infection is widely prevalent in China, and there is no efficient way to cure it. Antisense technique, blocking the target gene and inhibiting its expression at the molecular level, has shown potential antiviral and antitumor effects.［1-3］ Choosing the target site on the 3.2 kb HBV genome is the key to the antisense technique. In previous research, we found that an antisense oligonucleotide complementary to preS2 (as-preS2) had the strongest inhibitory effect on HBV antigen expression in vitro among four pieces of HBV antisense oligonucleotides. This fragment could induce apoptosis of hepatoma cells and lower telomerase activity.［4,5］ The findings demonstrated the therapeutic potential of as-preS2 against HBV chronic infection and HBV-related hepatocelluar carcinoma.
In this study, we assessed the specific expression of preS2 antisense RNA in hepatoma cells by a novel receptor-mediated gene transferring system and its inhibitory effect on nude mice bearing hepatocarcinoma.
METHODS
Plasmid
A hepatocarcinoma specific preS2 antisense RNA expression vector, pEBAF-as-preS2, was constructed in our lab. The HBV preS2 gene (3203-3340) was inserted into the reverse direction downward of the 5.5 kb of alpha-fetoprotein (AFP) promoter and enhancer ( Fig. 1 ). It could express antisense RNA against preS2 in AFP-positive cells.
Cells and their cultures
A hepatocelluar carcinoma cell line HepG2.2.15, integrated with 4 copies of the HBV genome, could not only accomplish the transcription and translation of HBV, but also produce HBV Dane's particles. The cell line was provided by Beijing Institute of Medicine and Biology and cultured in minimum essential medium (MEM) containing 10% foetal calf serum (FCS) and 380 μg/ml G418. The MEM was purchased from Hyclone Co., and the G418 and FCS from Gibco Co.
Animals
Bal B/C nude mice (male, 5-6 weeks of age) were purchased from the Experimental Animal Center of Tongji Medical College, Central China University of Science and Technology, China. Cells (1×107 of HepG2.2.15) were transplanted subcutaneously into the nude mice and were used when the tumor size reached 0.5-1 cm in diameter.
Synthesis of GE7-PL and HA20-PL
GE7, a 16-peptide specific to EGFR, and HA20, a homologue of the N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized by National Laboratory for Oncogenes and Related Genes, Shanghai Cancer Institute, China.［6］ GE7 and HA20 were conjugated with polylysine (PL), respectively （GE7-PL and HA20-PL.
Preparation of AFP-enhancing 4-element complex
pEBAF-as-preS2 DNA and GE7-PL, HA20-PL conjugates were independently mixed drop-wise into 10 μl of deionized water at different ratios. The mixture underwent reaction at 25℃ for 30 minutes. Mixture containing 0.2 μg plasmid DNA were analyzed with 1% agrose gel eletrophoresis to examine the retardation of DNA migration in order to determine the optimal ratio. According to the optimal ratio of DNA to conjugates based on the above experiment, a mixture of pEBAF-as-preS2 DNA and GE7-PL, HA20-PL conjugates was made to prepare the AFP-enhancing 4-element complex. After examining the retardation of DNA, the complex was used for gene transfection.
In vivo gene transfection targeting heptocellular carcinoma
AFP-enhancing 4-element complex containing 0.2 μg DNA was injected into the portal vein of nude mice bearing tumors. Animals were killed 3 days after injection, and total RNA was extracted from the tumor, heart, liver, spleen, kidney, and stomach according to TRIZOL kit protocol. According to the sequence described by Ono et al,［7］ the primers were designed to amplify the HBpreS2 gene: forward (S2P1): 5'-GCTCTAGACTCAGGCCATGCATG-3'; reverse (S2P2): 5'-GCTCTAGATGGTGAGTGATTGGAGGT-3'. All oligonucleotide primers were synthesized by the Shanghai Shenggong Bioengineering Company, China. Reverse transcription was performed in 20 μl volume including: 1 μg of total RNA, 4 μl of 5×buffer, 20 nmol/L dNTP, 20 units of RNAse inhibitor and 200 units of Moloney murine leukemia virus (M-MLV) reverse transcriptase. The reverse transcription reactions were performed at 42℃ for 1 hour, and then degenerated at 70℃ for 10 minutes to amplify the first strain of cDNA. The cDNA was used as templates for the amplification of preS2 with primers S2P1 and S2P2. PCR was carried out in 25 μl volume with 1 μl of cDNA, 2.5 μl of 10×buffer, 0.3 mmol/L Mg2+, 5 nmol/L dNTP, 3 units of Taq polymerase, and 10 nmol/L primers under fellow conditions: 94℃ for 5 minutes, then 35 cycles at 94℃ for 40 seconds, 50℃ for 40 seconds, 72℃ for 50 seconds, followed by a final extension period of 7 minutes at 72℃.
In vivo tumor-inhibitory experiments
Fifty-four nude mice bearing HepG2.2.15 were evenly divided into 6 groups randomly. In Groups A, B, and C, AFP-enhancing 4-element complex were injected into tumors of nude mice 15 days after inoculation of HepG2.2.15 cells (0.5 cm of mean tumor size). The doses of pEBAF-as-preS2 in AFP-enhancing 4-element complex were different in Groups A, B, and C, which were 0.2, 0.4 and 2 μg, respectively. The AFP-enhancing 4-element complex was given to each mouse once a week for four weeks. In Group D, AFP-enhancing 4-element complex containing 2 μg of pEBAF-as-preS2 was injected into tumors of nude mice 10 days after inoculation of HepG2.2.15 cells (0.2 cm of mean tumor size). Physiological salt solution (Group E) and empty vector (Group F) were given once a week for 4 weeks as controls. Tumor diameter was measured every 3 days. Animals were killed 5 weeks later and tumor masses were dissected and their diameters were measured.
RESULTS
Construction of AFP-enhancing 4-element complex
Polypeptide and plasmid were mixed at different ratios. According to the results of eletrophoresis in 1% agrose gel, DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1∶1. The AFP-enhancing 4-element complex was constructed successfully.
Hepatocarcinoma expression of antisense RNA mediated by AFP-enhancing 4-element complex
AFP-enhancing 4-element complex containing 0.2 μg pEBAF-as-preS2 DNA was injected into the portal vein of nude mice bearing tumors. Animals were killed 3 days later. Total RNA was isolated from the tumors, heart, liver, spleen, kidney, and stomach. Products were quantified by Biophometer (Eppendorf Co., country?). Total RNA ratio of A260/A280 from different tissues was 1.7-2.0. RT-PCR was carried out with specific primers (S2P1, S2P2) for the HBpreS2 gene. Two-percent gel eletrophoresis demonstrated that the products of amplification could be detected only in tumor tissues of animals treated with AFP-enhancing 4-element complex. No specific bands could be tested in other tissues ( Fig. 2 ).
Inhibitory effects of preS2 antisense RNA mediated by AFP-enhancing 4-element complex on tumor growth
After injection of AFP-enhancing 4-element complex containing 0.2 μg DNA into nude mice bearing tumor for 4 times, the mean diameter of tumor tissue was significantly shorter than that of the control (Groups E and F) (0.995±0.35 cm vs 2.125±0.25 cm, P<0.01). There was no significant difference between Groups E (1.97±0.37 cm) and F (the value in group F?, P>0.05).
Inhibitory effects of antisense RNA on tumors related to antisense RNA dosage
preS2 antisense RNA was given with doses of 0.2 μg (Group A), 0.4 μg (Group B), and 2 μg (Group C), respectively, once a week for 4 weeks. The diameter of tumor tissue in Group A was much shorter than that in Groups B or C, P<0.01 (Table).
Inhibitory effects of antisense RNA on tumorrelated to tumor size
In Group D, when the diameters of tumor were about 0.2 cm, the mice were treated with 2 μg DNA . Five weeks later, the tumor diameter was only 0.625±0.175 cm, significantly shorter than that of Group A (0.995±0.35 cm) in which 2 μg of DNA was given to each mouse once a week for 4 weeks from when the tumor diameter was about 0.5 cm.
DISCUSSION
There are three crucial factors in determining the efficacy of antisense gene therapy:［8］ first, whether the target gene plays a crucial role in tumorogenesis; second, whether the antisense RNA could be transfected uniquely to target cells; and third, whether it could be expressed efficiently and specifically in the targeted cells. In the present study, all these three factors were achieved by AFP-enhancing 4-element complex.
The HBV preS2 protein encoded by the preS2 gene has been shown to conjugate human serum albumin receptors and plays a key role in the HBV infection.［9,10］ It also has a trans-acting factor which can activate the expression of oncogenes and cell cycle regulating genes by binding DNA.［11,12］ Our previous study showed that antisense oligodeoxynucleotide against preS2（3203-3219）can not only decrease the expression of the HBV gene, but also suppress tumorgenesis in HepG2.2.15 cells.［4,5］ In the present study, in AFP-enhancing 4-element complex, the potential key gene, preS2, which might play an important role in regulating HBV expression and replication, was selected to be blocked. Inhibitory experiments in vitro had demonstrated that a block of preS2 gene (3203-3240) could efficiently inhibit the expression of HBV antigen in HepG2.2.15 cells (data not shown). In order to accomplish the directed delivery and transcription of targeted genes, virus expression vector containing hepatocarcinoma-specific regulatory sequences (AFP promoter) and an EGFR mediated tumor-specific gene delivery system were used in AFP-enhancing 4-element complex. In 1998, Tian et al［6］ established a novel high-efficiency receptor-mediated gene delivery system in which GE7 targeted on EGFR was used to transfer genes to tumor cells. The study implied that this system has a significant potential in gene therapy.［13］ However, the expression of EGFR is not limited to tumor cells. We have demonstrated that the green fluorescence protein (GFP) reporter gene could be also expressed in the epithelial cells of stomach mucosa and spermaduct after being transfected with 4-element complex in vivo (data not shown). In AFP-enhancing 4-element complex, a highly targetable transcription-regulatory sequence, AFP promoter, was used to build a hepatocarcinoma tissue-specific antisense RNA expression vector and direct the therapeutic gene to express specifically in AFP-positive hepatocellular carcinoma cells. RT-PCR indicated that AFP-enhancing 4-element complex could mediate preS2 antisense RNA specific expression in hepatoma cells of mice bearing tumors.
The merits of antisense RNA lie in its long-term, stable expression in vivo. To investigate the in vivo effects of pEBAF-as-preS2 on hepatomas, nude mice bearing hepatocellular carcinoma cell HepG2.2.15 were treated with different doses of AFP-enhancing 4-element complex. Results indicated that after four subcutaneous injections of AFP-enhancing 4-element complex containing 0.2 μg DNA, significant inhibition of tumor growth, as measured by tumor diameter, could be achieved. With higher doses of DNA (0.4 μg or 2 μg per mice), no corresponding increased inhibitory effect could be observed, implying that antisense RNA therapeutic effects can be achieved at low dosages with low prices.
The inhibitory effect of pEBAF-as-preS2 on tumors was also related to the administration time. Two μg of DNA injected once was enough to efficiently inhibit tumor growth when the diameter of the tumor was about 0.2 cm. When the diameter of the tumor was about 0.5 cm, four injections were required to achieve inhibitory effect. This shows that antisense therapy should be carried out at the early stage of tumor growth to achieve the best effects.
REFERENCES
1. Feng RH, Zhu ZG, Li JF, et al. Inhibition of human telomerase in MKN-45 cell line by antisende hTR expression vector induces cell apoptosis and growth arrest. World J Gastroenterol 2002;8:436-440.
2. Nie QH, Cheng YQ, Xie YM, et al. Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis. World J Gastroenterol 2001;7:363-369.
3. Wang XW, Yuan JH, Zhang RG, et al. Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice. World J Gastroenterol 2001;7:345-351.
4. Ma CH, Sun WS, Liu SX, et al. Comparision of the anti-HBV effect of various antisense oligodeoxynucleotides. Chin J Microbiol Immunl 2000:20;12-14.
5.Ma CH, Sun WS, Ding PF, et al. Inhibition of telomerase activity by preS2 antisense oligonucleotide and its role in inducing apoptosis of hepatocarcinoma cell. Chin J Microbiol Immunl 2000;20:461.
6. Tian PK, Ren SJ, Ren CC, et al. A novel receptor-targeted gene delivery system for gene thrapy. Sci Chin 1998;28:554-558.
7. Ono Y, Onda H, Sasaada R, et al. The complete nucleoide sequences of the cloned hepatitis B virus DNA: subtype adr and adw. Nucleic Acids Res 1983;11:1747- 1757.
8. Wang S, Dolnick Bj. Quantitative evalution of intracellular sense: antisense RNA hybrid duplexes. Nucleic Acids Res 1993;21:4383-4391.
9. Neurath AR, Strick N, Sproul P, et al. Search for hepatitis B virus cell receptors reveals binding sites for interleukin 6 on the virus enveloperprotein. J Exe Med 1992;175:146-151.
10. Kondo J, Shimomura H, Fujioka S, et al. Mutations in the hepatitis B virus preS2 region and abrogated receptor activity for polymerized human albumin. Acta Med Okayama 2002;56:193-198.
11. Alka S, Hemlata D, Vaishali C, et al. Hepatitis B virus surface (S) transactivator with DNA-binding properties. J Med Virol 2000;61:1-10.
12. Lauer U, Weiss L, Lipp M, et al. The hepatitis B virus preS2/St transactivator utilizes AP-1 and other transcription factors for transactivation. Hepatology 1994;19:23-31.
13. Lee TK, Han JS, Fan ST, et al. Gene delivery using a receptor-mediated gene transfer system targeted to hepatocellular carcinoma cells. Int J Cancer 2001;93:393-400.
|
PDF(-)
Rapid Response |